Just and inclusive end-of-life decision-making for long-term care home residents with dementia: a qualitative study protocol.

BACKGROUND: Many people living with dementia eventually require care services and spend the remainder of their lives in long-term care (LTC) homes. Yet, many residents with dementia do not receive coordinated, quality palliative care. The stigma associated with dementia leads to an assumption that people living in the advanced stages of dementia are unable to express their end-of-life needs. As a result, people with dementia have fewer choices and limited access to palliative care. The purpose of this paper is to describe the protocol for a qualitative study that explores end-of-life decision-making processes for LTC home residents with dementia. METHODS/DESIGN: This study is informed by two theoretical concepts. First, it draws on a relational model of citizenship. The model recognizes the pre-reflective dimensions of agency as fundamental to being human (irrespective of cognitive impairment) and thereby necessitates that we cultivate an environment that supports these dimensions. This study also draws from Smith's critical feminist lens to foreground the influence of gender relations in decision-making processes towards palliative care goals for people with dementia and reveal the discursive mediums of power that legitimize and sanction social relations. This study employs a critical ethnographic methodology. Through data collection strategies of interview, observation, and document review, this study examines decision-making for LTC home residents with dementia and their paid (LTC home workers) and unpaid (family members) care partners. DISCUSSION: This research will expose the embedded structures and organizational factors that shape relationships and interactions in decision-making. This study may reveal new ways to promote equitable decision-making towards palliative care goals for LTC home residents with dementia and their care partners and help to improve their access to palliative care.

Role of the intronic elements in the endogenous immunoglobulin heavy chain locus. Either the matrix attachment regions or the core enhancer is sufficient to maintain expression.

High level expression in mice of transgenes derived from the immunoglobulin heavy chain (IgH) locus requires both the core enhancer (Emu) and the matrix attachment regions (MARs) that flank Emu. The need for both elements implies that they each perform a different function in transcription. While it is generally assumed that expression of the endogenous IgH locus has similar requirements, it has been difficult to assess the role of these elements in expression of the endogenous heavy chain gene, because B cell development and IgH expression are strongly interdependent and also because the locus contains other redundant activating elements. We have previously described a gene-targeting approach in hybridoma cells that overcomes the redundancy problem to yield a stable cell line in which expression of the IgH locus depends strongly on elements in the MAR-Emu-MAR segment. Using this system, we have found that expression of the endogenous mu gene persists at substantially (approximately 50%) normal levels in recombinants which retain either the MARs or Emu. That is, despite the dissimilar biochemical activities of these two elements, either one is sufficient to maintain high level expression of the endogenous locus. These findings suggest new models for how the enhancer and MARs might collaborate in the initiation or maintenance of transcription.

Structural and functional analysis of J chain-deficient IgM.

Previous studies have discerned two forms of polymeric mouse IgM: moderately cytolytic (complement-activating) pentamer, which contains J chain, and highly cytolytic hexamer, which lacks J chain. To investigate the relationships among polymeric structure, J chain content, and cytolytic activity, we produced IgM in J chain-deficient and J chain-proficient mouse hybridoma cell lines. Both hexamer and pentamer were produced in the absence as well as the presence of J chain. Hexameric IgM activated (guinea pig) complement approximately 100-fold more efficiently than did J chain-deficient pentamer, which, in turn, was more active than J chain-containing pentamer. These results are consistent with the hypothesis that J chain-containing pentamer cannot activate complement. We also analyzed the structure of IgM-S337, in which the mu-chain bears the C337S substitution. Like normal IgM, IgM-S337 was formed as a hexamer and as both J chain deficient- and J chain-containing pentamers. Unlike normal IgM, IgM-S337 dissociated in SDS into various subunits. For IgM-S337 pentamer, the predominant subunits migrated as mu2kappa2 and mu4kappa4, and the subunit distribution was unaltered by J chain. However, J chain was found only in the mu2kappa2 species, suggesting that some arrangement of inter-mu bonds directs incorporation of J chain. IgM-S337 hexamer also dissociated to mu2kappa2 and mu4kappa4, but also yielded several species migrating much more slowly in SDS-PAGE than wild-type mu12kappa12. To account for these forms, we propose that each mu-chain can interact with three other mu-chains and that some hexameric molecules contain two catenated mu6kappa6 circles.

Interplay of J chain and disulfide bonding in assembly of polymeric IgM.

Normal mouse IgM is synthesized as hexamers in the absence of J chain and as pentamers in its presence. Previous work has suggested that polymer size is also closely related to formation of the inter-mu chain disulfide bond mediated by cysteine 414, one of three cysteines involved in inter-mu chain bonding. This correlation in turn suggested that formation of C414-C414 might be required for J chain to influence how IgM assembles and that formation of C414-C414 might affect the J chain/IgM stoichiometry. To test such hypotheses we have used cell lines which either expressed or did not express J chain to produce IgM in which serine was substituted for C414. In contrast to the case of IgM assembled from normal mu chains, IgM-S414 was secreted mostly as pentamers and tetramers but not as hexamers, irrespective of J chain synthesis. These results indicate that the role of J chain as modulator of IgM structure and function requires C414. Moreover, a more detailed analysis of the structure of J-plus and J-minus IgM-S414 revealed that J chain, in fact, influenced the nature of secreted IgM-S414: In the absence of J chain, some IgM-S414 was secreted as dimers and trimers, while in the presence of J chain, some IgM was secreted as non-covalently assembled pentamers. These results imply that disulfide bonding can occur differently from the pattern depicted in conventional models of IgM structure.

Analysis of IgM structures involved in J chain incorporation.

J chain is associated with pentameric IgM and polymeric IgA. In IgM, J chain is disulfide bonded to the C575 residue of the mu-chain, located in the mu tail piece (mu tp). Previous studies indicated that mu tp is not sufficient to mediate J chain incorporation into polymeric Ig. In this study, we analyzed which other C mu domains are involved in J chain incorporation. Three altered forms of mouse IgM were analyzed: IgM lacking the C mu 1 domain, IgM in which the C mu 2 and C mu 3 domains were replaced by the hinge region and the C gamma 2 domain of IgG2b, and IgM, in which the C mu 4 domain was replaced by C gamma 3. We found that neither C mu 1, C mu 2, nor C mu 3 was absolutely required for J chain incorporation. The importance of C mu 4 could not be fully analyzed because the C gamma 3 replacement mutant failed to form polymers. Also, we found that the glycosylation site at asparagine 563 of mu tp was important for J chain incorporation. Disruption of this site by replacement of either asparagine 563 by tyrosine or serine 565 by phenylalanine resulted in diminished J chain incorporation and increased production of hexameric IgM. These results demonstrate the importance of structural elements located close to mu tp in the incorporation of J chain into IgM.

Assembly of IgM. Role of disulfide bonding and noncovalent interactions.

Polymeric IgM is usually envisaged as an array of mu 2L2 monomers in which the mu heavy chains are held together by disulfide bonds involving cysteines at positions 337, 414, and 575. We have studied the importance of inter-mu-chain disulfide bonds for formation of IgM polymers and monomers by analyzing the effects of eliminating one or more of these disulfide bondings. Ablation of all inter-chain bonds by either chemical reduction and alkylation or by mutagenesis resulted in the exclusive production of halfmers (muL) molecules. IgM composed of mu-chains bearing each of the other six possible combinations of cysteine to serine replacements was produced as different mixtures of polymers, monomers, and halfmers. Cysteine 575 was both necessary and sufficient for efficient assembly of IgM polymers and sufficient but not necessary for efficient assembly of monomers. Cysteine 337 was sufficient but not necessary for efficient assembly of monomers, and was neither sufficient nor necessary for formation of polymers. Cysteine 414 was neither necessary nor sufficient for efficient formation of either monomers or polymers. Data also suggest that noncovalent interactions between C mu 2 domains are stronger than the interactions between C mu 4/tail domains.

On the linkage between RNA processing and RNA translatability.

The immunoglobulin mu heavy chain gene of mouse hybridoma cells is expressed in two forms, microseconds and microns, differing in their use of 3' exons. As for many other mammalian genes, mutations in the mu gene which prematurely terminate translation often have the effect of reducing the amount of these mu RNAs. To test the generality of this relationship, we selected mutant hybridoma cell lines defective in IgM production and searched both for translation termination mutations which do not reduce the amount of mu RNA as well as for mutants which show the more commonly observed reduction in mu RNA. As observed previously, the amount of microseconds RNA is normal in mutants terminating in the C mu 4 exon; by contrast the amount of microns RNA is reduced in these mutants, indicating that the effect of the mutation is influenced by some feature near the 3' end of the RNA. Mutations terminating translation in other C region exons have a graded effect on RNA content, ranging from 10% the normal level for termination in the C mu 3 exon down to 1% for termination in the C mu 2 exon. By contrast, a mutant cell line terminating in the leader exon contained 25% the normal amount of mu RNA, suggesting that translation past some point might be required to fully engage the RNA degradation process.

Enhancement of the antibody response to protein antigens by specific IgG under different experimental conditions.

Specific stimulation of the antibody response to protein antigens by IgG antibodies was studied in vivo. The response to TNP-coupled keyhole limpet haemocyanin (KLH-TNP) was enhanced by a TNP-specific IgG monoclonal antibody, 7B4, as measured both in ELISA (enzyme-linked immunosorbent assay) and by ELISPOT (enzyme-linked immunospot) method. Enhancement was seen when using both high and low doses of antigen and antibody. The antibody response to TNP-coupled bovine serum albumin (BSA-TNP), but not to TNP-coupled ovalbumin (OA-TNP), tetanus toxoid (TT-TNP) or diphtheria toxoid (DT-TNP), was also efficiently augmented by 7B4. Enhancement was seen when using both high and low coupling ratio of TNP to KLH. The fact that IgG-mediated enhancement is seen under many different experimental conditions suggests that it is a physiologically relevant phenomenon. In order to enhance the response to KLH-TNP and BSA-TNP, 7B4 had to be injected while antigen was circulating in blood. This supports that IgG-mediated stimulation acts by concentrating circulating antigen into lymphoid centres.

Inhibition of immunological memory and T-independent humoral responses by monoclonal antibodies specific for murine complement receptors.

The importance of the complement system for mounting an antibody response in vivo was investigated by down-regulating and blocking the complement receptors (CR) in mice with three different monoclonal rat antibodies (mAb): mAb 8C12 recognized the C3b-binding site of CR1, mAb 7G6 recognized another site of CR1 and the C3d-binding site of CR2 and mAb 7E9 again recognized other epitopes on CR1 and CR2. We have earlier shown that 7G6, is the only mAb that completely suppresses the primary antibody response to horse erythrocytes. This antibody was also shown to suppress induction of immunological memory and a secondary antibody response. In contrast to what seems to be the case for thymus-dependent antibody responses, all three mAb could inhibit the antibody response to a thymus-independent antigen, dextran B 1355S.

Evidence of IgG-mediated enhancement of the antibody response in vivo without complement activation via the classical pathway.

The complement (C) dependency of IgG-mediated enhancement of the antibody response was investigated by immunizing mice with trinitrophenyl-coupled keyhole limpet hemocyanin (TNP-KLH) and either a C-activating TNP-specific monoclonal IgG2a antibody (Hy-1.2) or a mutant, non-C-activating variant of Hy-1.2 (M12). Hy-1.2 as well as M12 efficiently enhanced the anti-KLH response, although Hy-1.2 was more active. In addition, also a naturally non-C-activating TNP-specific IgG1 antibody enhanced the response to TNP-coupled bovine serum albumin. Moreover, C-activating IgG could enhance the antibody response in mice depleted of C3 by treatment with cobra venom factor. These findings suggest that the classical pathway of C activation is not required for IgG-mediated enhancement.

In vivo inhibition of the antibody response by a complement receptor-specific monoclonal antibody.

BALB/c mice were injected intravenously with three different monoclonal antibodies (mAbs) specific for complement receptor 1 (CR1). Two of the mAbs crossreacted with CR2. 24 h later, the mice were immunized with horse erythrocytes or keyhole limpet hemocyanin (KLH), and the primary antibody response was measured. One of the anti-CR antibodies, 7G6, suppressed greater than 99% of the direct plaque-forming cell response against horse red blood cells (HRBC). The same antibody markedly suppressed the serum antibody responses to both HRBC and KLH. To be optimally suppressive, the mAb had to be injected before suboptimal concentrations of antigen. The other two complement receptor-specific antibodies had very moderate, if any, effects on the antibody response. 7G6 was able to downregulate CR1 and CR2 on the surface of B cells and, in addition, to inhibit rosette formation with C3d-coated sheep erythrocytes (EC3d). One of the antibodies with a weak effect downregulated only CR1. The other downregulated both CR1 and CR2, although not as efficiently as 7G6, and was unable to inhibit EC3d rosette formation. We conclude that the reason 7G6 is outstanding in its suppressive capacity is that it is the only mAb tested that functionally blocks CR2. The data suggest that CR2 is of crucial importance in the initiation of a normal antibody response to physiological concentrations of antigen.

Dual immunoregulatory effects of monoclonal IgG-antibodies: suppression and enhancement of the antibody response.

Nine monoclonal IgG-anti-TNP antibodies were investigated for their ability to modulate anticarrier responses in mice immunized with sheep red blood cells-2,4,6-trinitrophenyl (SRBC-TNP) or keyhole limpet haemocyanin-TNP (KLH-TNP). The antibodies enhanced the anticarrier response when KLH-TNP was used as antigen but suppressed it when SRBC-TNP was used. The enhancing and suppressive effects were not exerted by entirely the same sets of antibodies. The suppression was correlated to efficient antigen binding, but not complement activation, haemagglutination, or isotype of the monoclonal antibodies. In contrast, enhancement was correlated to isotype and complement activation but not to antigen binding capacity. Both the enhancing and the suppressive effects seem to require Fc-mediated functions of the IgG molecules since they modulate the anti-carrier response although they recognize hapten determinants. Thus, one and the same monoclonal hapten-specific IgG-antibody can enhance the anti-KLH response up to 38-fold whereas it suppresses the anti-SRBC response by more that 10-fold.

Complement activation is not required for IgG-mediated suppression of the antibody response.

Feedback suppression of the antibody response by IgG is known to be dependent on intact Fc regions. However, it is not clear which of the Fc-mediated effector functions is required. In the present report we have studied whether ability or inability of the IgG antibodies to activate the complement system was of consequence for their immunosuppressive effect. First, a monoclonal IgG1-anti-2,4,6-trinitrophenyl (TNP) antibody, unable to activate complement via the classical or alternate pathway, was shown to be able to inhibit more than 90% of the in vivo sheep erythrocyte-specific antibody response in mice when TNP coupled to sheep erythrocytes was used as antigen. Second, we investigated the immunosuppressive ability of a non-complement-activating mutant IgG2a-anti-TNP monoclonal antibody. The mutant differs from the wild type by a single amino acid substitution in the CH2 domain leading to inability to fix complement factor C1q. However, the mutant has the same affinity for antigen and the same Fc receptor-binding capacity as the wild type antibody. It is demonstrated that the mutant was as efficient as the wild type antibody in inhibiting an in vitro antibody response to TNP-coupled sheep erythrocytes. These findings confirm the non-determinant specificity and Fc dependence of IgG-mediated suppression, and show that the Fc-mediated effector mechanism is independent of complement activation. The results instead suggest binding to Fc receptors as a necessary step in feedback immunosuppression and favor inactivation of B cells by cross-linking of Fc and antigen receptors on their surface rather than elimination of antigen by complement-dependent phagocytosis as the effector mechanism.

De novo calcification in lymph node metastasis.

Calcification in malignant lymphoma or in lymph node metastases from a malignant tumor are most often encountered after radiotherapy or chemotherapy. De novo calcifications in malignant lymphoma however, are very rare. As far as we know, de novo calcification in retroperitoneal lymph node metastases from carcinoma have not been described before. A case is presented and the literature is reviewed.