Restoration of lymphocyte function in Janus kinase 3-deficient mice by retroviral-mediated gene transfer.

Janus kinase-3 (JAK3) deficiency has recently been identified as a cause of severe combined immunodeficiency (SCID) in humans. We used a mouse model of Jak3-deficient SCID to test a gene therapy approach for treatment of this disease. Transfer of a Jak3 retroviral vector to repopulating hematopoietic stem cells resulted in increased numbers of T and B lymphocytes, reversal of hypogammaglobulinemia, restoration of T-cell activation upon stimulation with mitogens, and development of an antigen-specific immune response after immunization. Analysis for vector copy number in lymphoid and myeloid populations showed a large in vivo selective advantage for Jak3-expressing lymphoid cells. These results show that gene replacement is a feasible treatment strategy for this disease and that naturally occurring in vivo selection of corrected cells is an important advantage of this approach.

Thymic lymphoproliferative disease after successful correction of CD40 ligand deficiency by gene transfer in mice.

Inherited deficiency of the CD40 ligand (X-linked hyper-IgM syndrome) is characterized by failure of immunoglobulin isotype switching and severe defects of cell-mediated immunity. To test the potential for gene transfer therapy to correct this disorder, we transduced murine bone marrow or thymic cells with a retroviral vector containing the cDNA for the murine CD40 ligand (CD40L) and injected them into CD40L-/- mice. Even low-level, constitutive expression of the transgene stimulated humoral and cellular immune functions in these mice. With extended follow-up, however, 12 of 19 treated mice developed T-lymphoproliferative disorders, ranging from polyclonal increases of lymphoblasts to overt monoclonal T-lymphoblastic lymphomas that involved multiple organs. Our findings show that constitutive (rather than tightly regulated), low-level expression of CD40L can produce abnormal proliferative responses in developing T lymphocytes, apparently through aberrant interaction between CD40L+ and TCRalphabeta+CD40+ thymocytes. Current methods of gene therapy may prove inappropriate for disorders involving highly regulated genes in essential positions in proliferative cascades.

A modified stereotaxic device for small bird anaesthesia and neurosurgery.

This report describes simple modifications to a stereotaxic instrument that allow for the administration of an inhalant anaesthetic and provide improved access to avian dorsal forebrain structures along with a more natural orientation for small birds during neurosurgery.

Genetic control of host resistance to flavivirus infection in animals.

Flaviviruses are small, enveloped RNA viruses which are generally transmitted by arthropods to animals and man. Although flaviviruses cause important diseases in domestic animals and man, flaviviral infection of animals which constitute the normal vertebrate reservoir may be mild or sub-clinical, which suggests that some adaptation between virus and host may have occurred. While this possibility is difficult to study in wild animals, extensive studies using laboratory mice have demonstrated the existence of innate, flavivirus-specific resistance. Resistance is heritable and is attributable to the gene Flvr, which is located on chromosome 5 in this species. The mechanism of resistance is at present unknown, but acts early and limits the replication of flaviviruses in cells. While some evidence supports a role for Flvr in enhancing the production of defective interfering virus, thereby restricting the production of infectious virus, other reports suggest that Flvr interferes with either virus RNA replication or RNA packaging. Recent research suggests that cytoplasmic proteins bind to the viral replication complex and that allelic forms of these proteins in resistant mice may restrict the production of infectious progeny. Apparent resistance to flaviviruses has been described in other vertebrates, although it remains to be seen if this is attributable to a homologue of Flvr. Nonetheless, knowledge gained of the characteristics and function of Flvr in mice should be applicable to other host species, and improvement of resistance to flaviviral infection in domestic animals by selective breeding or gene technology may ultimately be possible.

Treatment of frozen shoulder by manipulation under anaesthetic and injection: does the timing of treatment affect the outcome?

The effect of timing of a manipulation under anaesthetic (MUA) and injection of corticosteroid and local anaesthetic for the treatment of frozen shoulder has attracted little attention to date. All studies describe a period of conservative treatment before proceeding to an MUA. Delay has been associated with a poorer outcome. We present a retrospective review of a prospectively collected, single-surgeon, consecutive series of 246 patients with a primary frozen shoulder treated by MUA within four weeks of presentation. The mean duration of presenting symptoms was 28 weeks (6 to 156), and time to initial post-operative assessment was 26 days (5 to 126). The Oxford shoulder score (OSS) improved by a mean of 16 points (Wilcoxon signed-ranks test, p < 0.001) with a mean OSS at this time of 43 (7 to 48). Linear regression analysis showed no correlation between the duration of presenting symptoms and OSS at initial follow-up (R2 < 0.001) or peri-operative change in OSS (R2 < 0.001) or OSS at long-term follow-up (R2 < 0.03). Further analysis at a mean of 42 months (8 to 127) revealed a sustained improvement with a mean OSS of 44 (16 to 48). A good outcome follows an MUA and injection of corticosteroid and local anaesthetic in patients with primary frozen shoulder, independent of the duration of the presenting symptoms, and this improvement is maintained in the long term.

Phenotypic inhibition of the renin-angiotensin system, emergence of the Ren-2 gene, and adaptive radiation of mice.

The renin-angiotensin system in sub-genus Mus displays unique features including duplication of the renin gene in most strains, strong expression of the second gene in submandibular gland of males, and inhibited responses to injected renin. Our findings indicate that this inhibition results from a paucity of renin substrate and is consistent with first-order kinetics. We find substrate paucity to be a feature of both sexes and all sub-species and strains of Mus irrespective of gene duplication. Attempts to increase the level of substrate in blood by intravenous injection caused marked increases in blood pressure in Mus, suggesting that substrate paucity was a phenotypic prerequisite for successful emergence of enhanced renin expression in salivary gland. We propose that these phenomena are linked to salivary "lethal factor", possibly transferred by biting, in an evolutionary sequence that has provided a major selective advantage for Mus and influenced the ecology and evolution of rodents.

Distinctive kinetics of the antibody-forming cell response to Sendai virus infection of mice in different anatomical compartments.

The single-cell ELISPOT assay was used to determine the frequency and isotype commitment of virus-specific antibody-forming cells (AFC) at different anatomical locations following intranasal Sendai virus infection of C57BL/6 and 129/Sv mice. AFC responses in the mediastinal and cervical lymph nodes showed sharp increases and declines, first of IgM AFC, peaking about 7 days after infection, and then of IgG and IgA AFC, peaking about 10 days after infection. A wave of IgM AFC preceding the other isotypes was less evident in the spleen, where peak frequencies of AFC occurred 14 days after infection. Virus-specific AFC appeared in the bone marrow with a unique kinetic pattern, increasing in frequency gradually over the first 3 weeks after infection to a plateau that remained constant. Circulating IgM and IgG achieved significant titers approximately a week after infection; IgM titers were transient, but IgG levels increased sharply and remained high, reflecting the longevity of the bone marrow AFC response. Strain differences in isotype bias were noted, particularly preferential switching to the gamma 2a gene in 129/Sv mice. The B-cell response to acute respiratory viral infection thus exhibits features that are distinct from the primary response to nonreplicating antigens.

Respiratory virus infection of mice provokes a permanent humoral immune response.

We have observed that respiratory virus infection of mice provokes an extremely persistent humoral immune reaction, due to a long-sustained population of antibody-secreting cells in the bone marrow. Theories of humoral immunity that strongly distinguish primary and secondary reactions thus may not adequately describe the immune response to respiratory viruses.

Factors influencing palliative care. Qualitative study of family physicians' practices.

OBJECTIVE: To examine factors that influence family physicians' decisions to practise palliative care. DESIGN: Qualitative method of in-depth interviews. SETTING: Southwestern Ontario. PARTICIPANTS: Family physicians who practise palliative care on a full-time basis, who practise on a part-time basis, or who have retired from active involvement in palliative care. METHOD: Eleven in-depth interviews were conducted to explore factors that influence family physicians' decisions to practise palliative care and factors that sustain their interest in palliative care. All interviews were audiotaped and transcribed verbatim. The analysis strategy used a phenomenological approach and occurred concurrently rather than sequentially. All interview transcriptions were read independently by the researchers, who then compared and combined their analyses. Final analysis involved examining all interviews collectively, thus permitting relationships between and among central themes to emerge. MAIN OUTCOME FINDINGS: The overriding theme was a common philosophy of palliative care focusing on acceptance of death, whole person care, compassion, communication, and teamwork. Participants' philosophies were shaped by their education and by professional and personal experiences. In addition, participants articulated personal and systemic factors currently affecting their practice of palliative care. CONCLUSIONS: Participants observed that primary care physicians should be responsible for their patients' palliative care within the context of interdisciplinary teams. For medical students to be knowledgeable and sensitive to the needs of dying patients, palliative care should be given higher priority in the curriculum. Finally, participants argued compellingly for transferring the philosophy of palliative care to the overall practice of medicine.

Genetically determined resistance to flavivirus infection in wild Mus musculus domesticus and other taxonomic groups in the genus Mus.

Inherited resistance to flaviviruses in laboratory mice is a rare trait conferred by an autosomal dominant gene (Flvr). To provide information on genetic resistance to flaviviruses in wild mice, we analysed (i) wild M. m. domesticus trapped in Australia, and (ii) mice representing other species and subspecies in the genus Mus. Mice were screened for resistance relative to C3H/HeJ mice by intracerebral challenge with Murray Valley encephalitis virus or yellow fever virus, and breeding studies were undertaken to identify inherited resistance factors. Widespread flavivirus resistance was demonstrated in Australian M. m. domesticus. A single, autosomal dominant Flvr-like gene appeared to be primarily responsible, but there was some evidence for additional inherited resistance factors. Flavivirus resistance was also identified in other taxonomic groups, and a genetic basis for this resistance was demonstrated in M. m. musculus (Skive), M. spretus, and M. spicilegus. Interestingly, M. m. musculus (CZI-O) were more susceptible than C3H/HeJ mice. Our findings show that genetic resistance to flaviviruses is common in divergent taxonomic groups in the genus Mus, suggesting that the trait has an ancient evolutionary origin, but whether flavivirus resistance genes have an anti-viral role or serve some other function is unknown.

TH cells primed during influenza virus infection provide help for qualitatively distinct antibody responses to subsequent immunization.

The quality of the primary Ab-forming cell (AFC) response in cervical lymph nodes and mediastinal lymph nodes of mice to intranasal influenza virus was strongly influenced by viral replicative capacity. IgA secretors were prominent in the early AFC response to infectious virus in mediastinal lymph nodes, while IgG expression was more frequent among isotypically switched AFC in cervical lymph nodes of the same mice; this pattern was reversed in the response to inactivated virus. Influenza viruses A/Puerto Rico/8/34 (A/PR8) and A/X-31 share six of eight genome segments, differing only in hemagglutinin (H1 in A/PR8, H3 in A/X-31) and neuraminidase (N1 in A/PR8, N2 in A/X-31) genes. These viruses therefore elicit extensively cross-reactive TH populations, though their glycoproteins are serologically unrelated. Mice recovered from an A/X-31 infection thus mount a primary B cell response against A/PR8 glycoproteins, when challenged with the latter virus, though this response can call upon memory TH cells. To assess the impact of memory TH populations on a primary Ab response, we compared the AFC response to inactivated A/PR8 in naive mice and mice that had cleared an A/X-31 infection. A/X-31 immune mice mounted a more vigorous AFC response against A/PR8 H1 and N1 glycoproteins than naive animals, when immunized intranasally with inactivated A/PR8. However the distribution of isotypes among H1/N1-specific AFC in lymph nodes of A/X-31-primed mice resembled that of naive mice. Evidently, in this functional context, memory TH cells retained the ability to help Ab responses different in quality from that generated during their primary reaction.

Genetic studies of flavivirus resistance in inbred strains derived from wild mice: evidence for a new resistance allele at the flavivirus resistance locus (Flv).

Studies of genetic resistance to flavivirus infection in laboratory mice have led to the development of a single model in which resistance is conferred by an autosomal dominant gene designated Flvr. Because of evidence suggesting that wild mice carry virus resistance genes which are not present in laboratory mice, we compared flavivirus resistance in the inbred strains CASA/Rk, CAST/Ei, and MOLD/Rk, which are derived directly from wild mice, and the congenic strains C3H/RV (Flvr/Flvr) and C3H/HeJ (Flvs/Flvs). Resistance to the Murray Valley encephalitis virus strain OR2 and the 17D vaccine strain of yellow fever virus was assessed by determining the lethality of intracerebral infection and by measuring virus replication in the brain. The resistance of the CASA/Rk and CAST/Ei strains resembled the resistance of C3H/RV mice, whereas the resistance of the MOLD/Rk strain was intermediate between those of C3H/RV and C3H/HeJ mice. Genetic analyses showed that resistance in both the CASA/Rk and MOLD/Rk strains is conferred by single autosomal dominant alleles at the Flv locus. Our data indicate that flavivirus resistance in the CASA/Rk strain is due to a gene which is similar or identical to Flvr, whereas resistance in the MOLD/Rk strain is due to a previously undescribed gene which we designate Flvmr to indicate minor resistance to flavivirus infection. Since genetic resistance to flaviviruses is rare in laboratory mice, the CASA/Rk and MOLD/Rk strains will be valuable for further investigation of this phenomenon.

Differential antigen burden modulates the gamma interferon but not the immunoglobulin response in mice that vary in susceptibility to Sendai virus pneumonia.

Sendai virus, a paramyxovirus which causes murine pneumonia, grew to approximately 10-fold higher titers and was cleared less rapidly from the lungs of 129/J (129) than H-2b-compatible C57BL/6J (B6) mice. The more susceptible 129 mice also made higher titers of gamma interferon (IFN-gamma) and immunoglobulin G2a (IgG2a) virus-specific antibody. Analysis with acutely irradiated (950 rads) mice and immunologically reconstituted bone marrow (BM) radiation chimeras indicated that the enhanced virus growth was a function of the radiation-resistant respiratory epithelium. Prolonged exposure to more virus in turn influenced the magnitude of IFN-gamma production, most of which was made by CD4+ T lymphocytes. Somewhat surprisingly, however, the 129 pattern of a higher virus-specific serum Ig response skewed towards IgG2a mapped to the reconstituting BM. Thus, the characteristics of the humoral response are at least partly dissociated from both the antigen load, resulting from viral replication, and the level of IFN-gamma production. Further analysis of double chimeras (B6+129 BM-->B6 recipients) confirmed that the divergent humoral immune response to Sendai virus in B6 and 129 mice is largely determined by the inherent characteristics of the lymphoid cells.

Mapping the Flv locus controlling resistance to flaviviruses on mouse chromosome 5.

Genetically determined resistance to flaviviruses in mice is a dominant trait conferred by alleles at a single autosomal locus designated Flv, but no gene products have been associated with this locus and the mechanism of resistance is not well understood. To further characterize this model of genetic resistance, we conducted mapping studies to determine the chromosomal location of Flv. Because of evidence suggesting that the Flv locus is on chromosome 5, three-point backcross linkage analyses were used to define the location of Flv relative to previously assigned chromosome 5 markers. The results confirm the chromosome 5 location of Flv and indicate a map position between the anchor loci rd and Gus-s. The chromosomal localization of Flv is the first step in the production of a detailed linkage map of the Flv region, which may open approaches to positional cloning of the resistance gene.

Administration of anti-IFN-gamma antibody to beta 2-microglobulin-deficient mice delays influenza virus clearance but does not switch the response to a T helper cell 2 phenotype.

Treatment of mice that were homozygous for a beta 2-microglobulin gene disruption with a mAb that was specific for IFN-gamma delayed clearance of an influenza A virus from the respiratory tract for at least 3 days, whereas administration of an anti-IL-4 mAb had no effect. However, all mice survived and eventually cleared the virus. The anti-IFN-gamma significantly decreased both the level of class II MHC glycoprotein expression and the numbers of CD4+ lymphocytes in the inflammatory populations recovered by bronchoalveolar lavage of the pneumonic lung, whereas the total cell counts remained the same. These differences were not apparent for the regional mediastinal lymph nodes, although the frequency of lymph node B cells producing virus-specific Ab of the IgG2a subclass was greatly reduced. However, neither the anti-IFN-gamma nor anti-IL-4 treatments drastically altered the cytokine production profiles detected for freshly isolated lymphocytes by the using single cell ELISPOT assay or by ELISA of culture supernatants after in vitro restimulation with virus. Thus, neutralization of secreted IFN-gamma during the course of an influenza-specific response in beta 2-microglobulin-deficient mice that lack CD8+ T cells delays virus clearance and modifies the character of the host response, but does not cause the CD4+ subset to switch to a Th2 cytokine profile.

Evidence on patient-doctor communication.

This chapter covers important and well-studied aspects of patient-doctor communication. First the paper describes the lessons learned from studies about patients' satisfactions or dissatisfactions related to patient-doctor communication, making the point that complaints about doctors are usually due to communication problems and not technical competency issues. The next section of the chapter deals with time. It is often assumed that effective communication is inefficient. While this is not necessarily the case, the research results are complex and very interesting. The third part of the chapter covers communication in relation to patient adherence with the management plan recommended by the doctor. There is strong evidence that communication affects patient adherence and that there are four key aspects of communication that can enhance the patients' co-operation with the management plan. The final topic is patients' health. Twenty-two studies indicate the generally positive effect of key dimensions of communication on actual patient health outcomes such as pain, recovery from symptom, anxiety, functional status, and physiologic measures of blood pressure and blood glucose.

Matching antibody class with pathogen type and portal of entry: cognate mechanisms regulate local isotype expression patterns in lymph nodes draining the respiratory tract of mice inoculated with respiratory viruses, according to virus replication competence and site of inoculation.

Intranasal deposition of Sendai virus (SV) in C57BL/6 mice provokes an Ab-forming cell (AFC) reaction in mediastinal (MLN) and cervical lymph nodes (CLN), which drain the lungs and upper respiratory tract, respectively. While the majority of AFC elicited by infectious SV at both sites produced IgG, the CLN response to SV rendered inactive in replication was restricted almost entirely to IgA, although isotype switching in mediastinal continued to be skewed heavily to IgG. However, in vitro restimulation of the accompanying virus-specific T cell populations from the two sites did not reveal any significant difference in lymphokine output, and isotype expression was not altered substantially in mice lacking IL-4 or IL-6 genes. To dissociate the response to specific Ags from the inflammatory reaction to viral infection, we examined the response to inactivated SV in the face of infection with influenza virus A/HKx31. The magnitude and IgA dominance of the anti-SV AFC population in the CLN were unaffected by a simultaneous, vigorous, IgG-dominated CLN anti-influenza reaction. Evidently, the characteristics of this antiviral response are determined primarily by cognate interactions. Moreover, the IgA bias of the CLN AFC response to inactivated SV was observed only when the virus was delivered intranasally: injection under the epidermis of the cheek, a site that has a lymphatic drainage into the CLN, resulted in an IgG-dominated CLN AFC reaction, lacking IgA. The site of deposition of a vaccine can thus have more influence on the pattern of isotypes induced than the site at which the immune response is initiated.

Human parainfluenza virus type 1 immunization of infant mice protects from subsequent Sendai virus infection.

Human parainfluenza virus type 1 (hPIV-1) infections are a common cause of "croup" and hospitalizations among young children, yet no vaccine is yet available. Sendai virus (mouse PIV-1) is the closest known homologue of hPIV-1. Here we address the possibility of using a xenotropic, nonpathogenic PIV as a vaccine in infants, by assessing the efficacy of hPIV-1 vaccination of infant mice against a subsequent challenge with Sendai virus. hPIV-1 was administered intranasally to mice age 3-6 days and shown by serum antibody ELISA and elispot analysis to elicit virus-specific IgM and isotype-switched antibody-forming cells (AFC). The response was completely cross-reactive between hPIV-1 and Sendai virus. Mice were challenged with Sendai virus 6-8 weeks later and generated AFC and serum antibody responses composed of IgM, as well as IgG and IgA, unlike challenged, age-matched controls. The high IgM response among AFC was not seen in mice primed as adults with hPIV-1 and challenged with Sendai virus. The hPIV-1 priming of infant mice afforded protection, as the majority of these mice survived the lethal Sendai virus challenge, as did all adult primed animals. These data support the notion that the unmodified xenotropic Sendai virus might function effectively in human infants as a vaccine against hPIV-1.