[Decision-making and end of life care]. / Les déclarations de volontés en fin de vie. De la terminologie juridique à la communication médecin généraliste/patient.

Taboos surrounding the end of life and death slowly diminish. People talk more and more about when and how they want or do not want to die, and of what will become of their body after their passing. General practitioners usually accompany their patients in illness and health, but also when death comes near. It is expected from them that, within their relationship to their patients, they create the opportunity to address the patients' last wishes and possibly to formalize them. Three laws as well as the Deontology Code of the Medical Order mention the issue of the last wishes. What comes out is that for the general practitioner, the situation is not always transparent. This article seeks to clarify the definitions (juridical), their perceptions and respect. Furthermore, a proposal that is fully in the spirit of the law is made to simplify current procedures relating to anticipated statements about end of life's wishes.

[Decision-making processes and medical care at the end of life]. / Processus décisionnels et soins médicaux en fin de vie.

The Federal Authority, the Ministry of Public Health and Environment, charged the Ghent University and the Brussels Free University to produce a continuous recording tool of data's concerning the decision-making processes and the medical care at the end of life, after defining the present state of the art. This tool is built up from a glossary and a questionnaire made up of closed questions with a prospective part and a retrospective part, and leaving the possibility of comments. This questionnaire, first submit to experts and two ethic committees, was sent anonymously to a broad sample of doctors of which 193 answered. This study brings out important information on the application of the laws on palliative care, on the rights of the patient and on euthanasia; it would be advisable to organise in the future a further systematic recording of the end of life conditions throughout a standardized questionnaire whose first version is presented here.

[Euthanasia and other medical decisions concerning the end of life in Belgium: epidemiologic studies]. / Euthanasie et autres décisions médicales concernant la fin de la vie en Flandre: étude épidémiologique.

BACKGROUND: The study presented here is the first replica of the Dutch death certificate study on end of life decisions (ELDs). The main objective was to assess the incidence of euthanasia (the administration of drugs with the explicit intention to shorten the patient's life at the explicit request of the patient), physician assisted suicide (PAS), and other ELDs in medical practice in Belgium (Flanders). METHODS: A 20% random sample of 3,999 deaths was selected from all death certificates between January 1 and April 30, 1998. The physicians who signed the death certificates received one mail questionnaire per death case. FINDINGS: The response rate of the physicians was 52%. The results were corrected for the non response bias, and extrapolated to estimated annual incidences after seasonal adjustment for causes of death. It was estimated that 1.3% (1.0-1.6%, CI: 95) of all deaths resulted from euthanasia or PAS. In 3.2% (2.7-3.8%, CI: 95) of all cases, the physician ended the patient's life with lethal drugs without the explicit request of the patient. Alleviation of pain and symptoms with opioids in doses with a potential life shortening effect preceded death in 18.5% (17.3-19.7%, CI: 95) of cases and nontreatment decisions in 16.4% (15.3-17.5%, CI: 95) of cases, of which 5.8% (5.1-6.5%, CI: 95) with the explicit intention of ending the patient's life. INTERPRETATION: ELDs are prominent in medical practice in Belgium (Flanders). The incidence of deaths preceded by an ELD is similar to the Netherlands, but greater than in Australia. However, in Belgium (Flanders) the incidence of ending of patient's life without the patient's explicit request (3.2%, 2.7-3.8% CI: 95) is similar to Australia (3.5%, 2.7-4.3% CI: 95), but significantly higher than in the Netherlands (0.7%, 0.5-0.9% CI: 95).

Marrow-derived CD40-positive cells are required for mice to clear Cryptosporidium parvum infection.

To clear a Cryptosporidium parvum infection, mice need CD4+ T cells, major histocompatibility complex class II, and an intact CD40-CD154 signaling pathway. CD40 is constitutively expressed on marrow-derived cells such as dendritic cells and B lymphocytes and is induced by gamma interferon (IFN-gamma) on most somatic cells. To determine whether the CD40 needed to clear a C. parvum infection has to be on marrow-derived mononuclear cells or on the epithelial cells that normally harbor the parasite, we transplanted CD40-/- mice with CD40+/- bone marrow and then infected them with C. parvum. These chimeras cleared the C. parvum infection, while CD40+/- controls transplanted with CD40-/- marrow cells remained infected. CD40 expression on marrow-derived cells therefore suffices for a C. parvum infection to be cleared, while CD40 expression on intestinal epithelial cells is not sufficient. There was no difference between the acquisition of CD69 and CD154 by mesenteric lymph node T cells of C. parvum-infected animals with intact or disrupted CD40-CD154 pathways. CD4 T cells entered the intestinal laminae propriae of C. parvum-infected animals whether or not the CD40 genes of these recipients were intact. These results suggest that, for a C. parvum infection to be cleared, CD40 is not necessary for T-cell activation but may instead contribute to an effector pathway of marrow-derived cells.

End-of-life decisions in medical practice in Flanders, Belgium: a nationwide survey.

BACKGROUND: Our study is a repeat of the Dutch death-certificate study on end-of-life decisions (ELDs). The main objective was to estimate the frequency of euthanasia (the administration of lethal drugs with the explicit intention of shortening the patient's life at the patient's explicit request), physician-assisted suicide (PAS), and other ELDs in medical practice in Flanders, Belgium. METHODS: A 20% random sample of 3999 deaths was selected from all deaths recorded between Jan 1 and April 30, 1998. The physicians who signed the corresponding death certificates received one questionnaire by post per death. FINDINGS: The physicians' response rate was 1355 (52%). 1925 deaths were described. The results were corrected for non-response bias, and extrapolated to estimated annual rates after seasonal adjustment for death causes, and we estimate that 705 (1.3%, 95% CI 1.0-1.6) deaths resulted from euthanasia or PAS. In 1796 (3.2%, 2.7-3.8) cases, lethal drugs were given without the explicit request of the patient. Alleviation of pain and symptoms with opioids in doses with a potential life-shortening effect preceded death in 10,416 (18.5%, 17.3-19.7) cases and non-treatment decisions in 9218 (16.4%, 15.3-17.5) cases, of which 3261 (5.8%, 5.1-6.5) with the explicit intention of ending the patient's life. INTERPRETATION: ELDs are prominent in medical practice in Flanders. The frequency of deaths preceded by an ELD is similar to that in the Netherlands, but lower than that in Australia. However, in Flanders the rate of administration of lethal drugs to patients without their explicit request is similar to Australia, and significantly higher than that in the Netherlands.

Interferon-gamma is required for innate immunity to Cryptosporidium parvum in mice.

Although CD4 T cells are required for recovery from cryptosporidial infection, mice with severe combined immunodeficiency (SCID) remain infected for long periods without ill effect. In contrast, mice whose ability to use interferon(IFN)-gamma is impaired, by neutralization or gene knockout, experience heavy cryptosporidial infection that may lead to death. To determine whether the innate immunity of SCID mice to Cryptosporidium parvum (CP) requires IFN-gamma, doubly immunodeficient C57BL/6 SCID-IFN-gamma knockout mice were bred. These mice experienced heavy CP infections of the gut; a significantly greater number became moribund or died, compared with mice carrying the SCID mutation alone or carrying disrupted IFN-gamma genes alone. Mice with gene disruptions of inducible nitric oxide synthetase or Fas/Fas ligand recovered normally from CP infection. The results indicate that mice unable to produce specific immune responses because of the SCID mutation require IFN-gamma to avoid death after infection with CP.

Eradication of Cryptosporidium parvum infection by mice with ovalbumin-specific T cells.

CD154 is necessary for mice to clear a Cryptosporidium parvum infection, but whether this ligand has to be expressed on T cells with specificity for C. parvum has not been determined. We infected DO11.10 (ovalbumin specific) T-cell receptor transgenic mice that had been bred to a RAG(-/-) background with C. parvum and found that the infection was cleared within 6 weeks, while RAG(-/-) controls were unable to clear C. parvum infection. Recovery was accompanied by an increase in the number of splenic T cells with the CD44(high) phenotype that characterizes memory cells. To determine whether a C. parvum-infected environment sufficed to activate transgenic T cells, we reconstituted C. parvum-infected BALB/c SCID mice with DO11.10 RAG(-/-) splenocytes. Fecal excretion of C. parvum antigen ceased in the 12 weeks following the adoptive transfer, unless the mice were also injected with tolerizing doses of ovalbumin. DO11.10 T cells were found in the submucosa of C. parvum-infected, but not uninfected, BALB/c SCID hosts within 48 h of injection. The transferred DO11.10 T cells divided and acquired a CD44(high) memory phenotype in C. parvum-infected, but not uninfected, recipients. DO11.10 splenocytes from CD154 knockout donors failed to clear a C. parvum infection, confirming a requirement for CD154 in recovery. In vitro, the DO11.10 cells did not proliferate in response to C. parvum antigen, and a tBlast GenBank search revealed no matches between the ovalbumin peptide and C. parvum DNA sequences. C. parvum-infected SCID mice given RAG(-/-) CD8(+) T cells with a Listeria-specific transgene did not recover from C. parvum infection. Our data suggest that antigen-nonspecific CD4(+) T-cell effector mechanisms in combination with the innate arm of the immune system are sufficient for the eradication of C. parvum infection.

Liver and bile duct pathology following Cryptosporidium parvum infection of immunodeficient mice.

Patients with acquired immune deficiency syndrome (AIDS) and boys with mutations of the CD154 gene (causing congenital X-linked immunodeficiency with hyper-IgM [XHIM]) are susceptible to chronic infections of the biliary tract with Cryptosporidium parvum (CP) that may lead to biliary sclerosis and ultimately to cholangiocarcinoma. To determine whether the CP infection and the consequent immune response contribute independently to this morbidity, we infected mice with severe combined immunodeficiency (SCID) or with disrupted genes for CD154, CD40, or interferon gamma (IFN-gamma) with CP. Even when CP infection persisted for 16 weeks, the SCID mice developed only mild triaditis, without apoptosis of biliary epithelial cells (BEC). Fifty percent of the CD154 knockout mice developed lobular hepatitis with acute and chronic triaditis. The CD40 knockout mice developed marked triaditis, and the IFN-gamma knockouts either succumbed to enteritis or survived to develop marked triaditis, portal fibrosis, biliary sclerosis, necrosis with dilation of duct-like structures within the porta hepatis, and dysplastic changes. CP-infected SCID mice reconstituted with T cells from IFN-gamma knockout donors either developed severe enteritis or survived to develop triaditis, cholangitis, lobular hepatitis with periductular sclerosis, and scarring. Mice with disruptions of both the CD40 and IFN-gamma genes remained infected by CP and developed bile duct and liver disease, but not enteritis. Our results suggest that T-cell cytokines are required for the inflammatory and sclerosing responses to CP infection in immunodeficient animals. The response of immunodeficient mice to CP infection may model at least the initial steps toward the development of sclerosing cholangitis or bile duct cancers in XHIM patients.

Cytokine production in varicella-zoster virus-stimulated cultures of human blood lymphocytes.

Estimates of responder cell frequency (RCF) based on limiting dilution analyses are laborious, and alternative means to quantitate cell-mediated immunity to immunogens are desirable. It was shown that levels of interleukin (IL)-2 in the supernatant of varicella-zoster virus-stimulated blood lymphocytes from immune adults peaked at 48 h of culture and correlated partially with estimates of RCF (r = .74, P = .003). Levels of gamma-interferon, IL-4, and IL-10 increased through the first 4 days of culture, and gamma-interferon levels showed some correlation with peak IL-2 levels (r = .48, P = .03). Nevertheless, correlations between levels of these cytokines and RCF did not reach statistically significant levels.

Requirement of CD40-CD40 ligand interaction for elimination of Cryptosporidium parvum from mice.

Mice with disrupted genes for CD40 and CD40 ligand (CD40L) are unable to clear infection with Cryptosporidium parvum and develop cholangitis. Parasites are present in the gut, gall bladder, and biliary tree, and biliary epithelial cells express CD40 on the cell surface. SCID mice infected with C. parvum for >1 month can clear the infection after reconstitution with spleen cells from CD40, but not CD40L, knockout mice. In an in vitro model, C. parvum-infected HepG2 cells were triggered to apoptosis when incubated with a CD40L-CD8 fusion protein. The requirement for CD40-CD40L interactions for immunity to C. parvum indicated by our results may entail the triggering of apoptosis in infected cells, in addition to the known role of CD40L-CD40 interactions in stimulating cytokine production and promoting T-cell responses.

Cholangiopathy and tumors of the pancreas, liver, and biliary tree in boys with X-linked immunodeficiency with hyper-IgM.

We report an association between X-linked immunodeficiency with hyper-IgM (XHIM) and carcinomas affecting the liver, pancreas, biliary tree, and associated neuroectodermal endocrine cells. The tumors were fatal in eight of nine cases and in most instances were preceded by chronic cholangiopathy and/or cirrhosis. An additional group of subjects with XHIM had chronic inflammation of the liver or bile ducts but no malignancy. Many patients with XHIM were infected with cryptosporidia. CD40 is normally expressed on regenerating or inflammed bile duct epithelium. A CD40+ hepatocellular carcinoma cell line, HepG2, susceptible to cryptosporidia and CMV infection became resistant when cell surface CD40 was cross-linked by a CD40 ligand fusion protein. Apoptosis was triggered in HepG2 cells if protein synthesis was blocked by cycloheximide or if the cells were infected by cryptosporidia. Ligation of CD40 on biliary epithelium may contribute to defense against infection by intracellular pathogens. We propose that the CD40 ligand mutations that cause XHIM deprive the biliary epithelium of one line of defense against intracellular pathogens and that malignant transformation in the biliary tree follows chronic infection or inflammation. The resulting tumors may then progress without check by an effective immune response. Patients with XHIM who have abnormal liver function tests should be considered at increased risk for cholangiopathy or malignancy.

Frequency and cytokine phenotype of blood T cells from premature infants responding to staphylococcal enterotoxin B.

The responder cell frequency (RCF) of premature (< 1900 g birth weight) infants' blood lymphocytes, which proliferate in cultures stimulated by staphylococcal enterotoxin B, falls from 1:3400 to about 1:8000 during the first 2 wk of life. Term infants, in contrast, show no fall in RCF. The reduced RCF in the premature infants affected cells that make interferon-gamma more than cells making IL-4. The reduced RCF was accompanied by a fall in the fraction of V beta 3+ T cells that entered cell cycle in stimulated cultures. The RCF of premature infants' T cells was increased in cultures supplemented with irradiated monocytes from adults. Addition of IL-4 (but not IL-2, IL-6, or indomethacin) increased the RCF and fraction of cells entering cell cycle of the premature infants. The data suggest that postnatal environmental factors limit the ability of premature infants' monocytes to support a T-cell response to staphylococcal enterotoxin B in vitro and that this limitation is overcome by adding IL-4.

Cytokine production in varicella zoster virus-stimulated limiting dilution lymphocyte cultures.

Human blood lymphocytes were stimulated with varicella zoster virus (VZV) antigen in limiting dilution cultures and the amounts of interferon-gamma (IFN-gamma) and IL-4 measured in the supernatants. The results indicate that up to 85% of proliferating cells of young adults produce IFN-gamma and up to 10% make IL-4. At limiting dilution, few if any wells were positive for both IFN-gamma and IL-4. The amount of IFN-gamma per well increased in the presence of antibody to IL-4, but anti-IFN-gamma not increase IL-4 production. The frequency of wells containing IFN-gamma was lower in subjects < 19 or > 55 years of age, and the amounts of IFN-gamma in positive wells was significantly lower in cultures of the older subjects' lymphocytes. The frequency of IL-4-making cells did not fall significantly with age. The data suggest that the age-related decline in the frequency of blood T cells which responds to VZV affects mainly the cells with a Th1-like cytokine phenotype.

Proliferative and cytokine responses by human newborn T cells stimulated with staphylococcal enterotoxin B.

Staphylococcal enterotoxins are potentially valuable tools for investigating the development of T-cell responses because in experimental animals they can elicit either T-cell activation and proliferation or tolerance. Previous studies indicate that human T cells bearing the CD45RA phenotype (which account for the majority of newborn T cells) respond poorly to stimulation by staphylococcal enterotoxin B (SEB) compared with mature T cells from adult blood. The present studies show that the mean frequency of newborn T cells that proliferated in limiting dilution cultures stimulated by SEB was 1:3135, with a 1SD range of 3153-4191 compared with a mean of 1:493 and range of 120-1737 for adult T cells. Neither indomethacin nor the nitric oxide synthesis inhibitor, n-arginine methyl ester, increased SEB responses by newborn cells, arguing against down-regulation of the newborn response by prostaglandin or nitric oxide. Naive (CD45RA+) T cells from adult blood had a responder cell frequency to SEB similar to that of the newborn cells. IL-2 production by newborn cells was delayed compared with adult cells but was equivalent after 3 d of culture. Production of gamma-interferon and IL-4 was greater by adult than newborn cells. Our results indicate that a subset of CD45RA+ cells that is activated by SEB can mature to make IL-4 or gamma-interferon after 3-5 d. The limiting dilution assay results provide a quantitative basis for proliferation by naive T cells against which responses by T cells from healthy and premature newborns can be compared.

Herpes simplex virus interferes with monocyte accessory cell function.

HSV is a successful human pathogen that causes severe infections in immunocompromised adults and newborn humans. The possibility that HSV might evade host immune responses by interfering with accessory cell function was investigated in vitro using newborns' monocytes adhered to plastic. These cells lost their ability to present staphylococcal enterotoxin B to resting T cells in a stimulatory form after overnight culture with HSV. The interference with T cell proliferation required live virus and was abolished by heat- or UV inactivation. The T cell proliferative response was restored by the addition of IL-2. Furthermore, HSV-precultured monocytes had a reduced production of IL-1 alpha and TNF-beta after phorbol-ionomycin stimulation. Wild-type HSV and a HSV mutant lacking the primary virion host shutoff protein both interfered with IL-1 synthesis and presentation of staphylococcal enterotoxin B for T cell proliferation. These results suggest that HSV can interfere with the provision by human monocytes of costimulator factors that are essential for T cell stimulation. This effect of HSV may be due to secondary shutoff mechanisms that decrease host protein synthesis or secretion after HSV infection.

Varicella-zoster virus (VZV)-specific cytotoxicity after immunization of nonimmune adults with Oka strain attenuated VZV vaccine.

Cytotoxic, proliferative, and serum IgG antibody responses to varicella-zoster virus (VZV) were measured monthly in 29 young adults for 3 months following primary immunization with the Oka strain attenuated VZV vaccine. No subjects had lymphocytes stimulated to VZV-specific cytotoxicity at study entry although lymphocytes from 2 subjects proliferated when stimulated by VZV antigen. The percentages of subjects with positive major histocompatibility complex class II-restricted cytotoxic responses at 1, 2, and 3 months after immunization were 54%, 69%, and 66%, respectively. Correlations between cytotoxic, proliferative, and antibody responses were highest in the first month after immunization; lower but still statistically significant after the second month; and not apparent by the third month. The data suggest that antibody and cell-mediated immune responses to VZV immunization develop in parallel after immunization, but these effector mechanisms are independently regulated by 3 months after immunization.

T cell proliferative response to interleukin 2: different frequency of responders among CD45R0 and CD45RA subsets.

A proportion of human blood T cells is known to proliferate in cultures containing 10 or more units/ml of interleukin (IL)2, in the absence of exogenously added antigen. A possible explanation for this phenomenon is that the IL2 serves to maintain the proliferation of memory T cells which have recently been activated by antigen in vivo. To test this possibility we therefore separated T cells according to their CD45 (common leukocyte antigen) phenotype into the CD45RO and CD45RA subsets which are associated with memory and naive status, respectively. The frequency of responder cells in limiting dilution analysis of adult MNC was 1:1625 CD45RO cells, 1:2405 for CD45RA cells, and 1:1642 for unseparated cells. CD45RA cells from newborns had a 1:1850 frequency of responders. The IL2 responder cells were predominantly CD3+ TcR alpha beta +, with a minor component of TcR delta cells. There were no differences in the frequency of T cells using V beta 5 and V beta 8 between responding and nonresponding cells. The data argue against a requirement for prior sensitization for a proliferative response to IL2 and give no evidence that the subset of T cells which responds to IL2 differs in phenotype from nonresponsive cells.

V beta 5 and V beta 8 memory T cells in adults and infancy: co-ordinated increase in response to early antigen stimulus.

Blood mononuclear cells (MNC) expressing the T cell receptor V beta gene families were identified with the monoclonal antibodies 1C1 (V beta 5) or Mx6 (V beta 8). These cells were typed for expression of the T200 common leucocyte antigen with the CD45RA antibody defining the 220-kD variant (which characterizes naive cells) and the UCHL1 antibody which defines the 180-kD variant (characterizing memory cells). Of adult MNC, 3 +/- 0.44% stained for V beta 5 and 4.12 +/- 0.26% stained for V beta 8; similar results were obtained in a group of infants aged less than 3 months, who were exposed to antigen stimulus in the form of infection and/or blood transfusion. While the total percentages of V beta 5 and V beta 8 cells in the blood of adults were not correlated, the proportions of these cells expressing CD45RO was positively correlated. A similar trend which did not reach statistical significance was present in a group of infants. A minority of the infants studied showed transient deviations from the mean established for adults. The results suggest that naive and memory T cell populations have similar frequencies of V beta 5 and V beta 8 expressing cells, and that age does not affect the frequency of usage of either of these V beta families.