Two apparently unrelated groups of symbiotic annelids, Nautiliniellidae and Calamyzidae (Phyllodocida, Annelida), are a clade of derived chrysopetalid polychaetes.

Nautiliniellidae Miura and Laubier, 1989 is a small family of marine polychaetes with 20 currently described species in 11 genera, most of which are known to live symbiotically in the mantle cavity of bivalves, mainly from cold seeps and hydrothermal vents, while Calamyzidae (Hartmann-Schröder, 1971) including only one described species, Calamyzas amphictenicola Arwidsson 1932 lives as an ectoparasite on ampharetid polychaetes in Swedish waters. Nautiliniellidae and Calamyzidae have both been considered to belong to Phyllodocida, but the few phylogenetic studies including these taxa have found their positions unstable. The internal relationships within Nautiliniellidae are also poorly understood. Using molecular information from both nuclear and mitochondrial genes and morphological data we assessed the systematic placement of Nautiliniellidae (seven species; collected from Pacific hydrothermal vents and cold seeps and one from Atlantic waters) and Calamyzas amphictenicola. Our results show that C. amphictenicola and Nautiliniellidae formed a well-supported clade that is nested within Chrysopetalidae, a free-living group of polychaetes. The chrysopetalid genus Vigtorniella Kiseleva 1992; a bacterial mat grazer found at methane seeps, anoxic basins and whalefalls, formed a paraphyletic grade with respect to the Nautiliniellidae-Calamyzas clade. The internal relationships within the Nautiliniellidae-Calamyzas clade as well as the relationships with their hosts are also examined. As a result we synonymize Calamyzidae and Nautiliniellidae with Chrysopetalidae, with the last as the oldest available family-group name. Within Chrysopetalidae we refer to the subfamilies Chrysopetalinae Ehlers 1864; Dysponetinae Aguado, Nygren & Rouse, herein; and Calamyzinae Hartmann-Schröder, 1971. Calamyzinae contains C. amphictenicola, all taxa formerly in Nautiliniellidae, and the chrysopetalid genus Vigtorniella.

Technologies to improve immunisation safety.

Ever since a vaccine was first used against smallpox, adverse events following immunisation have been reported. Adverse reactions may be caused by a fault in vaccine production, idiosyncratic responses or unsafe handling and vaccine administration practices. Technological advances that promise to bypass many of the dangers currently associated with vaccine administration are described. Plans for the next decade and beyond include developing injection-free systems for vaccine delivery that overcome the limitations of current immunisation programmes and help prevent programmatic mistakes. Also under development are new parenteral administration devices such as the auto-disable syringe and the mono-dose pre-filled device, and mucosal and transcutaneous immunisation systems. Training needs to be at the forefront of efforts to limit human error. Above all, there must be a willingness to respond to new climates and new technologies in order to ensure safe immunisation of children globally.

Ensuring vaccine safety in immunization programmes--a WHO perspective.

Ever since vaccines were firstly used against smallpox, adverse events following immunization have been reported. As immunization programmes expand to reach even the most remote communities in the poorest countries, it is likely that many more events will be temporally linked with vaccine administration. Furthermore, the profound shift in the general public and media interest in adverse events may lead to undue concerns and allegations which may ultimately jeopardize immunization programmes world-wide. While the health professional has understood this issue for some time, the public and the media have now also become all too aware of the significance of vaccine-related adverse events. The familiar vaccines, well-tested over decades, have not changed--but the perception regarding their safety has shifted. Claims outrageous or reasonable are being made against both the old and the newly-introduced vaccines. At the same time, the immunological and genetic revolution of the last decade may well bring to our notice some hypothetical risks that need to be addressed at pre-clinical level. WHO has been at the leading edge to guarantee vaccine safety for the last 30 years and will continue to do so. The Organization's plans for the next decade and beyond include the Safe Injection Global Network (SIGN), the development and introduction of safer technologies, and the prevention, early detection and management of AEFIs. The new technologies include needle-containing injection devices such as the autodisable syringe, as well as mucosal and transcutaneous immunization. Training will continue to be at the centre of WHO's efforts, limiting human error to a minimum. Mechanisms have been set in place to detect and respond to new and unforeseen events occurring. Above all, there is a willingness to respond to new climates and new technologies so that the Organization is in the best position to ensure safe immunization for all the world's children.

Recombinant bacillus Calmette-Guérin as a potential vector for preventive HIV type 1 vaccines.

In August 1997, the World Health Organization (WHO) and the Joint United Nations Programme on HIV/AIDS (UNAIDS) convened an expert working group to discuss current strategies for the development of HIV type 1 vaccines. Based on the recent findings of investigators from Japan's National Institute of Infectious Diseases (NIID) in Tokyo using recombinant bacillus Calmette-Guérin (rBCG) as a potential vectored vaccine for HIV, a recommendation was made that further work in this area is a priority. As a result, the working group reconvened in September 1998 to discuss the progress to date with this vaccine approach, as well as areas of related research to assess the feasibility of a BCG-vectored HIV vaccine. This report summarizes the discussions addressing the available scientific data on the potential use of rBCG as a vector for preventive HIV vaccines, the work necessary to move such candidate vaccines into Phase 1 clinical trials, and recommendations targeted at facilitating the long-term development of rBCG-vectored HIV vaccines.

Future approaches to vaccine development: single-dose vaccines using controlled-release delivery systems.

The development of single-dose vaccines, mainly those administered during childhood, which would effectively protect against certain diseases, would be a very important advance towards better immunization coverage and protection against the respective pathogens. Biodegradable polymeric microspheres which are 'programmed' to deliver the antigen when a boost of the immune response is required, may be a possible way of achieving this goal.

Towards vaccine optimisation.

New molecular technologies have accelerated the search for sub-unit candidate vaccines. However, once identified the use of a candidate antigen must be optimised to reap the maximum benefit from the eventual vaccine. This optimisation should take into account both the needs of the target population, and the various ways of potentiating the protective immune response induced. One must be sure that the final product will be used. Hence, vaccine optimisation should strive toward meeting the needs of a specific epidemiological problem within the economic constraints of a given situation. This may be possible using novel delivery systems designed to limit the number of doses needed, improve the stability or facilitate the delivery of a particular vaccine. In meeting the needs of a target population in a field situation, one must also keep in mind certain safety factors that go beyond the usual regulatory constraints. The immune response to vaccine candidates can be potentiated in many ways. The ability to preferentially induce specific protective effector mechanisms: i.e., antibody isotypes, T-cell subsets, and T-cell sub-subsets, is becoming a reality. Carrier molecules designed to avoid the problems of epitope suppression and competition, and perhaps an eventual "carrier jam," are being developed. Adjuvants and immunostimulants may also help, but the critical issue here remains their acceptability for use in man. Finally novel strategies for the induction of the immune response may also potentiate the immune response in the optimisation of vaccines.

Human lung cancer--a comparative study of the levels of circulating immune complexes in pulmonary blood draining the tumor area and peripheral venous blood.

Our objective was to investigate whether low levels of circulating immune complexes (cICs) in peripheral venous blood of cancer patients could be due to removal of cIC released at the tumor site during passage to the peripheral veins. In 54 patients with primary lung cancer, we therefore compared the cIC levels as detected by 3 different assays in paired samples from the pulmonary vein draining the tumor area and from a peripheral vein. Only 6 of the 54 patients had significantly increased pulmonary vein cIC levels as compared to the corresponding peripheral vein levels. The peripheral vein levels of these 6 patients were all within the normal range, and in none of these patients was the difference between the 2 sites of analysis--although significant--of such a magnitude that the pulmonary vein cIC level appeared higher than the normal range, i.e., "positive" for cIC. Positive cIC levels were only found in 11% of lung cancer patients (irrespective of the site of measurement). Thus, our present data, together with our previous findings indicating no significant difference between peripheral venous blood cIC levels in cancer patients and normal controls, contradict the theory of tumor cells expressing new antigens resulting in the formation of tumor-associated cICs.

Genetic diversity of murine rheumatoid factors.

Anti-Ig autoantibodies (rheumatoid factors, RF) have been implicated in the pathogenesis of human and murine rheumatoid arthritis as well as in the regulation of normal immune responses. Their genetic origin, clonal diversity, and inducing agents, and the relatedness between RF associated with disease and those occurring under physiologic conditions are not well understood. In this study, the genetic and clonotypic origin of 34 monoclonal IgM RF-secreting hybridomas from arthritic MRL-lpr/lpr and nonarthritic MRL-+/+ and C57BL/6-lpr/lpr mice was examined by RNA hybridization. For this purpose, we used probes for 10 VH and 13 Vk gene families as well as all JH and Jk gene segments. The majority of hybridomas expressed distinct Ig gene segment patterns and, hence, were clonally unrelated. Overall, a variety of different V and J gene segments were expressed in the hybridoma panel, suggesting that a large number of distinct genetic elements participates in expression of RF-like activity. RF from arthritic mice expressed Vk messages from the overlapping Vk22 and Vk28 gene families more frequently than did those from nonarthritic mice. RF from autoimmune MRL mice, both arthritic MRL-lpr/lpr and nonarthritic MRL-+/+, showed skewed JH4 segment usage, whereas those from C57BL/6-lpr/lpr preferentially expressed JH2.

Primary lung cancer. A controlled study of preoperative and postoperative levels of circulating immune complexes.

Three complement-dependent and one rheumatoid factor-dependent immune complex assays were used to analyze the sera taken before surgery from 70 unselected and previously untreated lung cancer patients, from 30 of them after surgery, and from 31 healthy controls. Plasma levels of complement split product C3d were also analyzed. The levels of circulating immune complexes (cIC) and C3d were essentially the same in samples taken from lung cancer patients before surgery and from healthy controls. By the four immune complex assays, increased levels were found in 0 to 10% of the preoperative lung cancer patients compared to 3% of the healthy controls (not significant). The postsurgical tumor, lymph node, metastasis (pTNM) stage of the lung cancer was not reflected in the levels of cIC or C3d. Paired comparisons of the cIC and C3d levels before and after surgery did not show significant differences. Thus, we found no evidence for the occurrence of cancer-related cIC in lung cancer patients.

A prospective study of circulating immune complexes in patients with breast cancer.

Levels of circulating immune complex (cIC) and complement split product C3d were studied in 86 patients with breast cancer (BC), 22 patients with benign breast disease (BD), and 72 age- and sex-matched blood-bank donors (NC), using solid-phase Clq-protein A RIA, Clq-anti-IgG RIA, anti-C3d anti-IgG RIA, and polyclonal IgM-rheumatoid factor ELISA for clC detection. No significant differences in cIC and C3d levels were found between the groups. The incidence of raised cIC levels varied from 4.9 to 8.2% in the BC group and from 4.5 to 22.7% in the BD group in comparison with 2.9 to 3.0% in the NC group. Using the solid-phase polyclonal IgM-rheumatoid factor ELISA we found that the cIC levels of patients with stage-III cancer were significantly higher than those of patients with stage-I or stage-II cancer. However, the other tests showed no relationship to tumor burden. Likewise, an effect of mastectomy on the cIC levels was also only detectable by one of the assays, i.e., the post-mastectomy levels of cIC as measured by the solid-phase anti-C3d anti-IgG RIA were significantly lower than the pre-mastectomy levels. Serial analyses of cIC and C3d levels were performed pre-operatively, one month post-operatively and every 3 months during the first year after mastectomy in 46 of the patients. During a I-year observation period, 7 patients developed metastatic disease. The occurrence of metastatic disease was not, however, preceded by characteristic changes in serially determined cIC and C3d levels.

Prognostic value of serial determinations of circulating immune complex levels in malignant melanoma.

A total of 50 melanoma patients free of distant metastatic disease and 54 healthy controls were analyzed for circulating immune complexes (cIC) and complement split product (C3d), using solid-phase Clq-anti-IgG radioimmunoassay (RIA), Clq-protein A RIA, and anti-C3d anti-IgG RIA for cIC detection. No significant differences in cIC and C3d levels could be demonstrated between the controls and the 31 patients with primary malignant melanoma analyzed before surgery. To evaluate the prognostic value of serial measurements, samples from the 50 patients were taken at regular intervals for 4 to 27 months (median, 20 months). Surgery was the only treatment given. Significant changes in the cIC and C3d levels were defined by reference to the changes that occurred in 23 of the 54 healthy controls observed for a period of 6 to 55 months (median, 23 months). During the period of serial sampling, recurrent disease developed in 8 of the patients. In only 3 of these 8 patients (versus 10 of 42 patients without recurrence) did significant changes occur, and the changes occurred either at the same time or after the clinical diagnosis of recurrence. During the entire clinical observation period of 6 years, a total of 11 patients developed recurrences. Significant changes were only observed in 4 of these 11 patients versus 8 of 37 patients without recurrence. In conclusion, changes in cIC and/or C3d levels were not found to be indicative of early or long-term recurrence of malignant melanoma.

The genetic origin of murine lupus-associated autoantibodies.

Systemic lupus erythematosus and rheumatoid arthritis in human and murine systems are characterized by circulating autoantibodies and immune complex deposition in various organs causing tissue damage and disease. To define the molecular and clonotypic origin of these anti-self responses, and to determine whether abnormalities in Ig genes or somatic mechanisms generating autoantibody diversity may contribute to lupus etiology, we performed molecular analyses of the Ig germline gene organization and the Ig gene segments expressed in monoclonal autoantibodies from autoimmune mice. Comparative restriction fragment length polymorphism analysis of a large number of Ig gene loci from autoimmune and normal mice indicated that (a) lupus can develop in different Ig heavy and kappa light chain variable region gene haplotypes, and (b) the Ig germline genes in lupus mice might be normal. To determine whether autoantibodies are encoded by unique Ig gene segments present in the normal germline repertoire, but not expressed in exogenous responses, we compared nucleic acid sequences encoding lupus autoantibodies and antibodies against foreign antigens. Similar, and in some instances even identical, gene segments were expressed in both types of antibodies, indicating that anti-self and anti-foreign responses use the same, or at least an overlapping, germline gene repertoire. A large variety of Ig variable, diversity, and joining gene segments encoded these autoantibodies with different specificities. Hence, the overall murine lupus-associated anti-self response may be essentially unrestricted. Furthermore, limited evidence has been obtained that both germline genes and somatically mutated genes encode autospecificity, making gross abnormalities in mechanisms for somatic mutation of Ig variable genes unlikely.(ABSTRACT TRUNCATED AT 250 WORDS)

Specificity and molecular characteristics of monoclonal IgM rheumatoid factors from arthritic and non-arthritic mice.

Two-hundred twenty-four hybridomas secreting monoclonal IgM rheumatoid factor (hIgMRF) derived from MRL-lpr/lpr, MRL-+/+ and C57BL/6-lpr/lpr autoimmune mice were analyzed with regard to IgG subclass and domain specificity, and some for VH gene expression patterns. Among these mice, only MRL-lpr/lpr develop arthritis. Clonotypes specific for each of the four mouse IgG subclasses and clonotypes reacting with more than one IgG subclass were identified. Although each panel contained several clonotypes, the predominant one differed in each strain (MRL-lpr/lpr, anti-IgG2a; MRL-+/+, combined anti-IgG2a and 2b; C57BL/6-lpr/lpr, anti-IgG1 or combined anti-IgG1, 2a, and 3). The IgG domains recognized by these monoclonals were defined with mutant Ig carrying IgG1 heavy chains that lacked either the CH1 or CH3 domains, variant Ig carrying hybrid IgG2b-2a heavy chains, and IgG fragments. Inhibition of hIgMRF binding to IgG substrates by protein A was also assessed. Most determinants were assigned to the CH3 domain, but determinants in the hinge region, CH2 domain, and in some instances, even in the Fab portion, could also be identified. Hybridization of cytoplasmic RNA from 35 classes of diverse IgG subclass specificity with VH gene probes representing seven of the approximately 10 VH families (7183, S107, Q52, J558, J606, 36-60, X24) indicated that approximately 90% of these clones expressed VH genes belonging to the large J558 gene family. The results indicate that murine IgMRF are extremely heterogeneous in IgG subclass and domain specificities; the genetic background influences RF specificity characteristics that may relate to pathogenicity; and considering the complexity of the J558 VH gene family and reported RF heavy chain assignments to additional VH gene families, it appears that VH genes encoding RF are diverse.

Clearance kinetics and organ uptake of complement-solubilized immune complexes in mice.

C3-bearing immune complexes were prepared by in vitro solubilization of BSA-anti-BSA complexes at equivalence. Sucrose density gradient analyses showed a size-heterogeneous population of solubilized complexes with a range of 7S to greater than 29S and a peak at around 19S. The presence of C3bi was demonstrated by precipitation with antibodies to C3c and to C3d and by binding to conglutinin. Immune complexes solubilized in two and three times antigen excess were selected as controls due to their size similarities with complement-solubilized complexes. Blood clearance curves were very similar for C3-bearing complexes and controls. At 1 hr, the percentage of injected material remaining in the circulation for complement-solubilized and two and three times antigen excess complexes were 29.5 +/- 1.3, 30.9 +/- 1.7 and 26.1 +/- 2.7, respectively. Uptake by liver accounted for the majority of complement- and antigen-solubilized immune complexes removed from circulation. Although the uptake by the spleen was no more than one-tenth of the liver uptake, more complement-solubilized complexes than antigen-solubilized complexes were removed by this organ. The present data indicate that soluble immune complexes bearing C3 components and soluble immune complexes without C3 components, but of comparable size, are cleared from the circulation of mice at comparable rates. The mechanisms of clearance of these two populations of complexes, however, may differ.

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b.

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.

Association of lpr gene with graft-vs.-host disease-like syndrome.

Hemopoietic cells have been reciprocally transferred between two lines of mice (MRL lpr/lpr and MRL +/+) that are congenic, differing only at the lpr (lymphoproliferation) and possibly closely linked genes. The lpr strain develops a significantly more severe and fast-paced lupus-like syndrome than +/+ strain, along with a substantially larger lymphoid mass. The results showed that: (a) hemopoietic cells of such mice were sufficient to induce the respective disease phenotypes in lethally irradiated syngeneic recipients; (b) cells of MRL +/+ mice maturing in an MRL lpr/lpr environment essentially retained the disease-producing characteristics of the donor, i.e., they induced late-life lupus without lymphadenopathy; but (c) MRL lpr/lpr cells transferred into irradiated MRL +/+ recipients unexpectedly failed to induce the early-life severe lupus and lymphoid hyperplasia of the donor, instead they caused a severe wasting syndrome resembling, in many respects, graft-vs.-host disease (GVHD). This GVHD-like syndrome developed after transfer of MRL lpr/lpr fetal liver, bone marrow, or spleen cells, and was not abrogated by elimination of T cells from the inocula. Thymectomy of the MRL +/+ recipients retarded, but did not prevent, the wasting disease. The unidirectional nature of this disease suggests that the lpr mutation conferred either a structural or regulatory defect that interfered, blocked, or altered the expression or structure of certain lymphocyte antigen(s). As a result, the MRL +/+ cells that did express this antigen(s) were recognized as foreign, and stimulated a graft-vs.-host reaction. These findings may allow definition of a new kind of rejection phenomenon caused by non-H-2 products, and may extend our understanding of the means by which the lpr gene adversely affects lymphocyte regulation and homeostasis.

Induction of severe autoimmune disease in normal mice by simultaneous action of multiple immunostimulators.

Either of two immunostimulating factors (lpr, lipopolysaccharide) enhanced the pathogenic autoimmune responses of MRL/n mice, but the serologic and immunopathologic characteristics differed. In contrast, either factor acting alone, caused minimal immunopathology in normal mice, despite autoantibody induction. Combined immunostimulation, however, caused fatal glomerulonephritis in normal-background C57BL/6 mice. These results show the profound influence of the background genome on the effects of immunostimulating agents, and show that resistance to autoimmune disease in immunologically normal mice is not absolute.