Association of immune parameters with clinical outcome in stage III colon cancer: results of Southwest Oncology Group Protocol 9009

Levamisole (LMS), utilized in the adjuvant treatment of patients with stage III colon cancer, is immunomodulatory. To determine whether alterations in immune parameters before, during and after 12 months of 5FU/LMS therapy correlate with disease-free survival, 38 patients enrolled on Southwest Oncology Group (SWOG) protocol 8899 received extensive lymphocyte phenotypic analysis prior to therapy and 3, 6, 12 and 15 months after treatment initiation. The median follow-up of patients is 41 months. Significant increases in the proportion and total number of CD56⁺ natural killer cells were seen, starting at 3 months and continuing until 15 months (P < 0.001). Increases in the total numbers of cells expressing CD25 (interleukin-2 receptor), VLA4 and the combinations of CD4: CD45RA and CD4:CDw29 were not evident during therapy but were seen at 15 months (P < 0.05: CD25, CD4:CDw29, CD4:CD45RA; P < 0.001: VLA4). Low levels of CD8⁺ cells prior to treatment initiation and after 3 months of therapy correlated with early relapse within the first year of 5FU/LMS treatment. Patients who have remained disease-free (n = 22, median follow-up 45 months) demonstrated increases in the total numbers of CD8⁺, CD25⁺, CD56⁺, VLA4⁺, CD4: CDw29 and CD4:CD45RA cells, primarily at 15 months. In contrast, patients who relapsed had decreased numbers of CD8⁺, CD4:CDw29, CD4: CD45RA and VLA4⁺ cells and minimal increases in CD56⁺ and CD25⁺ cells. Statistically significant differences between the late-relapse group and the group remaining disease-free were seen for CD25⁺, CD4: CD45RA and CD4:CDw29 cells at the 15-month assay time (P = 0.0276, P = 0.0349, P = 0.0178 respectively). In conclusion, multiple alterations in lymphocyte phenotype, with increases in the proportion and total number of cells involved in cell-mediated immune responses, were seen during and especially following completion of therapy with 5FU/LMS. Many of these changes are significantly associated with clinical outcome and may be useful for risk stratification of stage III colon cancer patients following completion of adjuvant therapy. Received: 9 July 1999 / Accepted: 11 August 1999     

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila

A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM.     

Does a deletion in a virus-like particle protein have pleiotropic effects on the reproductive biology of a parasitoid wasp?

Endoparasitoid insects have evolved mechanisms to counteract host immune defences. At oviposition, the endoparasitoid Venturia canescens injects virus-like particles (VLPs) together with the egg that interfere with the immune system of the host. These prevent encapsulation of the parasitoid egg. It has been shown that the gene coding for one of the VLP proteins exists in two variants (called Repeat-Plus (RP) and Repeat-Minus (RM)). Previous observations suggested that these variants induce pleiotropic effects on the reproductive biology of the female resulting, in an impeded transfer of eggs from the ovarioles to the oviducts in RM females, and in alterations in the egg laying sequence. We show that RM females from another geographical locality do not exhibit any phenotypic alteration of the reproductive biology. By showing a lack of association between VLP alleles and the reproductive phenotypic characteristics in a given strain, our results do not support the hypothesis of pleiotropic effects. Conversely, the results suggest that different genes code for the VLPs and for the reproductive biology characteristics. Different phenomena such as linkage, the action of pathogens, etc. could explain the association between characters that is observed in some strains.     

Patterns of natural selection on size at metamorphosis in water frogs

Strategies for optimal metamorphosis are key adaptations in organisms with complex life cycles, and the components of the larval growth environment causing variation in this trait are well studied empirically and theoretically. However, when relating these findings to a broader evolutionary or ecological context, usually the following assumptions are made: (1) size at metamorphosis positively relates to future fitness, and (2) the larval growth environment affects fitness mainly through its effect on timing of and size at metamorphosis. These assumptions remain poorly tested, because data on postmetamorphic fitness components are still rare. We created variation in timing of and size at metamorphosis by manipulating larval competition, nonlethal presence of predators, pond drying, and onset of larval development, and measured the consequences for subsequent terrestrial survival and growth in 1564 individually marked water frogs (Rana lessonae and R. esculenta), raised in enclosures in their natural environment. Individuals metamorphosing at a large size had an increased chance of survival during the following terrestrial stage (mean linear selection gradient: 0.09), grew faster and were larger at maturity than individuals metamorphosing at smaller sizes. Late metamorphosing individuals had a lower survival rate (mean linear selection gradient: -0.03) and grew more slowly than early metamorphosing ones. We found these patterns to be consistent over the three years of the study and the two species, and the results did not depend on the nature of the larval growth manipulation. Furthermore, individuals did not compensate for a small size at metamorphosis by enhancing their postmetamorphic growth. Thus, we found simple relationships between larval growth and postmetamorphic fitness components, and support for this frequently made assumption. Our results suggest postmetamorphic selection for fast larval growth and provide a quantitative estimate for the water frog example.     

Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae)

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA₃-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA₃-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.     

Successful vaccination of BALB/c mice against human hookworm (Necator americanus): the immunological phenotype of the protective response

In this murine (BALB/c) model of necatoriasis, high levels of protection against challenge infection by Necator americanus larvae (n=300) were afforded by successive vaccinations at 14-day intervals, either subcutaneously or percutaneously, with gamma-irradiated N. americanus larvae (n=300). Percutaneous vaccination was significantly more effective than the subcutaneous route, with pulmonary larval burdens at 3 days post-infection being reduced by 97.8 vs. 89.3%, respectively, after three immunisations (P<0.05). No worms were recovered from the intestines of thrice vaccinated mice. Two percutaneous vaccinations also reduced worm burdens, by 57% in the lungs and 98% in the intestines; P<0.05. In vaccinated animals, lung pathology (mainly haemorrhage) following infection was greatly reduced compared with non-vaccinated animals. In vaccinated mice (but not in non-vaccinated mice) mast cells accumulated in the skin and were degranulated. RT-PCR analyses of mRNAs in the skin of vaccinated animals indicated increased expression of interleukin (IL)-4 relative to gamma-interferon (gamma-IFN). Lymphocytes from the axillary (skin-draining) lymph nodes of vaccinated mice, stimulated in vitro with concanavalin A, exhibited enhanced secretion of IL-4 protein and a higher IL-4/gamma-IFN protein ratio than lymphocytes from non-vaccinated animals. In vaccinated mice, levels of IgG1 and IgG3 (directed against larval excretory/secretory products) were elevated for the most part compared with those in non-vaccinated animals. These data demonstrate the successful vaccination of BALB/c mice against human hookworm infection and suggest that a localised Th2 response may be important for conferring protection against necatoriasis.     

Comparative analysis of natural killer cell activity, lymphoproliferation and lymphocyte surface antigen expression in nonhuman primates housed at the CIRMF Primate Center, Gabon

Six different species of nonhuman primates housed at the CIRMF Primate Center, cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), mandrills (Mandrillus sphinx), vervets (Cercopithecus aethiops pygerythrus), chimpanzees (Pan troglodyte) and baboons (Papio hamadryas), were evaluated for their natural killer cell activity and for the ability of their peripheral blood mononuclear cells to proliferate in response to known mitogens (concanavalin A, phytohemagglutinin and staphylococcal enterotoxin A) and to react with a panel of mouse monoclonal antibodies directed against human leukocyte surface antigens. Basic information on normal immune functions in these primates is important because of their use as experimental animal models for the study of human diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis, loiasis and malaria.     

Large-scale production of mouse phenotypes: the search for animal models for inherited diseases in humans

This paper is aimed principally at bioinformaticians and biologists as an introduction to recent advances in mouse mutagenesis, concentrating on genome-wide screens utilising the powerful mutagen N-ethyl-N-nitroso-urea (ENU). It contains a brief background to the underlying genetics as well as details of the practical aspects of organisation and data capture for such projects.     

The psychopathological phenotype of velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) is mostly associated with deletions of chromosome 22q11, and is thought to be characterized by an increased frequency of major psychiatric disorders. Sixteen patients adults with VCFS and psychiatric symptoms were evaluated using a semi-structured investigation of history, symptoms, signs and behaviour. All available data were used in consensus meetings to obtain a classifiable diagnostic category. In contrast to other reports, no categorical diagnosis could be established. Instead, a quite specific psychological, behavioural and psychopathological constellation emerged that should most adequately be denominated as a VCFS-psychiatric syndrome. It is concluded that VCFS is associated with a specific psychopathological syndrome.