The role of macrophages in the development of biliary injury in a lipopolysaccharide-aggravated hepatic ischaemia-reperfusion model.

INTRODUCTION: Endotoxins, in the form of lipopolysaccharides (LPS), are potent inducers of biliary injury. However the mechanism by which injury develops remains unclear. We hypothesized that hepatic macrophages are pivotal in the development of endotoxin-induced biliary injury and that no injury would occur in their absence. MATERIAL AND METHODS: Clodronate liposomes were used to deplete macrophages from the liver. Forty-eight rats were equally divided across six study groups: sham operation (sham), liposome treatment and sham operation (liposomes+sham), 1mg/kg LPS i.p. (LPS), liposome treatment and LPS administration (liposomes+LPS), hepatic ischaemia-reperfusion injury with LPS administration (IRI+LPS) and liposome treatment followed by IRI+LPS (liposomes+IRI+LPS). Following 6h of reperfusion, blood, bile, and liver tissue was collected for further analysis. Small bile duct injury was assessed, serum liver tests were performed and bile composition was evaluated. The permeability of the blood-biliary barrier (BBB) was assessed using intravenously administered horseradish peroxidase (HRP). RESULTS: The presence of hepatic macrophages was reduced by 90% in LPS and IRI+LPS groups pre-treated with clodronate liposomes (P<0.001). Severe small bile duct injury was not affected by macrophage depletion, and persisted in the liposomes+IRI+LPS group (50% of animals) and liposomes+LPS group (75% of animals). Likewise, BBB impairment persisted following macrophage depletion. LPS-induced elevation of the chemokine Mcp-1 in bile was not affected by macrophage depletion. CONCLUSIONS: Depletion of hepatic macrophages did not prevent development of biliary injury following LPS or LPS-enhanced IRI. Cholangiocyte activation rather than macrophage activation may underlie this injury. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

A cohort study in university students: investigation of risk factors for cytomegalovirus infection.

Little is known about the main routes of human cytomegalovirus (HCMV) transmission in young adult populations. This study investigated risk factors for HCMV transmission in young adults attending university over a 3-year period. Blood samples were tested for HCMV specific viral capsid antigen IgG by enzyme immunoassay. Being born in a developing country and having lived in Africa were associated with HCMV seropositivity at study onset. No risk factors were associated with HCMV seroconversion over the 3-year follow-up. In contrast to previous reports, sexual activity was not associated with HCMV seroprevalence or seroconversion.

Epstein-Barr virus, B cell lymphoproliferative disease, and SCID mice: modeling T cell immunotherapy in vivo.

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) arises in up to 10% of organ transplant recipients and is fatal in ∼50% of cases. PTLD can be modeled in SCID mice using EBV+ve human B lymphoblastoid cell lines (BLCLs), and the current study investigated intraperitoneal (ip) inoculation of such animals in experiments which assessed the effect of EBV-specific cytotoxic T lymphocytes (CTLs) and cytokines on PTLD growth. Ip transfer of one dose of autologous CTLs, or CD8-enriched T cells, into ip BLCL-inoculated animals significantly delayed tumor development (P = 0.001) and prevented tumor formation in a significant proportion (40%) of mice (P = 0.001). A combination of interleukin (IL)2, 7, and 15 conditioning of CTLs prior to ip injection significantly delayed ip BLCL-derived tumor formation in vivo when compared to CTLs expanded in vitro using only IL2 (P = 0.04) and prevented tumor outgrowth in a significant proportion (60%) of mice (P = 0.02). Daily ip IL2 dosing of ip CTL-inoculated mice significantly delayed tumor development in vivo (P = 0.004) and prevented tumor outgrowth in a significant proportion (78%) of mice (P = 0.02) when compared to animals dosed with vehicle only. In SCID mice, autologous CTLs, and CD8-enriched T cells, have significant capacity to hinder development of PTLD-like tumors. Whilst studies are needed to delineate the role of cytokine conditioning and CD4-enriched T cells, the results suggest that IL2 plays a key role in supporting CTL funtion in vivo.

Tumour necrosis factor gene polymorphism: a predictive factor for the development of post-transplant lymphoproliferative disease.

BACKGROUND: Epstein-Barr virus-positive post-transplant lymphoproliferative disease (PTLD) is a potentially lethal complication of iatrogenic immunosupression after transplantation. Predicting the development of PTLD allowing early and effective intervention is therefore of importance. Polymorphisms within cytokine genes are implicated in susceptibility to, and progression of, disease however the published data are often conflicting. We undertook investigation of polymorphic alleles within cytokine genes in PTLD and non-PTLD transplant cohorts to determine risk factors for disease. METHODS: SSP-PCR was used to analyse single nucleotide polymorphism within tumour necrosis factor (TNF)-alpha, interleukin- 1, -6, -10 and lymphotoxin-alpha genes. The TNF-alpha levels were measured by standard enzyme-linked immuno-absorbant assay. RESULTS: We show an association between variant alleles within the TNF-alpha promoter (-1031C (P=0.005)); -863A (P=0.0001) and TNF receptor I promoter regions (-201T (P=0.02)); -1135C (P=0.03) with the development of PTLD. We also show an association with TNF-alpha promoter haplotypes with haplotype-3 significantly increased (P=0.0001) and haplotype-1 decreased (P=0.02) in PTLD patients compared to transplant controls. Furthermore, we show a significant increase (P=0.02) in the level of TNF-alpha in PTLD patient plasma (range 0-97.97 pg ml(-1)) compared to transplant controls (0-8.147 pg ml(-1)), with the highest levels found in individuals carrying the variant alleles. CONCLUSION: We suggest that genetic variation within TNF-alpha loci and the level of plasma cytokine could be used as a predictive risk factor for the development of PTLD.

Regulatory T cell activity in primary and persistent Epstein-Barr virus infection.

Regulatory T cells (T(reg)) provide a balance to immune T cell activation thereby protecting the body from pathogen-induced immunopathology. Several persistent viruses induce T(reg) that subvert protective immune mechanisms and promote viral persistence. Epstein-Barr virus (EBV) generally infects children subclinically and persists thereafter, but primary infection in early adulthood may cause immunopathological damage manifest as infectious mononucleosis. In this study the role of T(reg) was investigated in acute infectious mononucleosis and healthy EBV seropositive donors. The proportion of CD4(+)CD25(high) T cells in blood from infectious mononucleosis patients was significantly lower than in seropositive donors (P = 0.05). Using the FOXP3 marker for T(reg) the same frequency and extra-follicular distribution of T(reg) was noted in infectious mononucleosis and control tonsils. Regulatory cytokines, interleukin (IL)-10 and transforming growth factor (TGF)-beta, were significantly raised in infectious mononucleosis compared to seropositive donor plasma (P = 0.0001, P = 0.0004 respectively) although levels of IL-10 peaked earlier in infectious mononucleosis than TGF-beta. Previous studies identified EBV latent membrane protein (LMP)-1-induced T(reg) activity [Marshall et al. (2003): J Immunol 170:6183-6189; Marshall et al. (2007): Brit J Haematol 139:81-89], and in this study a significant reduction in interferon-gamma production was found from infectious mononucleosis but not seropositive donor lymphocytes after stimulation with a recall antigen when LMP-1 peptide PRG was added (P = 0.03). It is possible that T(reg) are important in controlling primary EBV infection to a subclinical level in most cases and that infectious mononucleosis represents a failure of this protective mechanism.

Patient-focused outcomes following detection in a hospital-based screening programme for C282Y haemochromatosis.

BACKGROUND: Haemochromatosis is a common genetic disease in populations of a northern European origin. However, there is uncertainty as to whether it is a condition that should be screened for. AIMS: To determine the proportion of persons, in a public hospital setting, who were homozygous for the C282Y mutation for hereditary haemochromatosis and the proportion of these persons who would benefit from therapeutic phlebotomy. METHODS: All persons who had blood submitted for pathology testing, had total iron-binding capacity and iron measured and transferrin saturation calculated, and where this result exceeded 40%, genotyping for the C282Y mutation was carried out. RESULTS: Of 18,779 patients screened, 887 (5.4%) were found to have transferrin saturation greater than 40%. Thirty-five of these were homozygous for the C282Y mutation. Fourteen were previously known to be affected and six of these were non-compliant with venesection. Venesection was commenced in 5 of the 21 newly diagnosed subjects. CONCLUSIONS: The proportion of detected subjects who commenced venesection was significant. Results suggest that clinical penetrance is higher in Australia than other countries and that even in the environment of a large tertiary teaching hospital, phenotypic screening identifies cases of hereditary haemochromatosis, which are likely to benefit from treatment.

Epstein-Barr virus latent membrane protein does not inhibit differentiation and induces tumorigenicity of human epithelial cells.

Latent membrane protein (LMP) is a latent Epstein-Barr virus (EBV) protein expressed in the EBV associated malignancy, nasopharyngeal carcinoma (NPC). Properties ascribed to this protein include inhibition of epithelial cell differentiation and deregulation of epithelial cellular gene expression, and are believed to contribute to the development of NPC. Studies to evaluate the oncogenic potential of LMP in epithelial cells have not been conclusive. We carried out studies to determine the tumorigenic activity of LMP in two human epithelial cell lines, SCC12F and HaCaT; while SCC12F LMP transfectants were non-tumorigenic in severe combined immunodeficient mice, HaCaT LMP transfectants were strongly oncogenic. The tumours produced were well differentiated, keratinising squamous cell carcinomas suggesting that LMP does not inhibit epithelial cell differentiation which conflicts with a previous report by Dawson et al. (1990). To resolve this discrepancy we examined the ability of HaCaT and SCC12F LMP transfectants to differentiate in a suspension culture assay. Both lines were able to differentiate to a similar extent as parental lines and control transfectants. Our results indicate that LMP is strongly oncogenic in human epithelial cells but that inhibition of differentiation is not necessarily a mechanism by which LMP contributes to the pathogenesis of NPC.

The ins and outs of EBV infection.

Epstein-Barr virus (EBV) infects almost the entire adult population of the world. The success of this virus appears to be based on its ability to infect the B cell, rather than any other cell type. We review EBV B-cell tropism, and discuss the mechanisms by which the virus may gain access to, and egress from, B cells in the normal host.

Infection of leukaemic B lymphocytes by Epstein Barr virus.

Epstein-Barr virus (EBV) infection of B lymphocytes in vitro gives rise to immortalized lymphoblastoid cell lines. Previous reports have shown that chronic lymphocytic leukaemia (CLL) cells, although infectable by EBV, are resistant to immortalization (1-4), although a small number of CLL cell lines have been reported (5-7). In the present study we have analysed early events occurring after EBV infection in 16 CLL samples. Out of 16 samples, 15 could be infected by the virus and expressed the full EB viral nuclear antigen (EBNA) complex but only one out of 16 expressed the latent membrane protein (LMP). The five CLLs in which we could investigate the presence of viral episomes showed circularized EBV by 16 hours after infection. The sequence of EBNA expression and genome circularization mirrored that seen in normal B cells, although genome amplification was not detected. The only CLL sample which expressed LMP after EBV infection was induced to proliferate for 2-3 weeks, but no cell line was established. Immortalized cell lines were obtained from three out of 16 samples tested, but all were polyclonal for light chain expression and had arisen from the CD5-negative, normal B-cell population. Thus the inability of EBV to induce proliferation of most CLL cells correlated with the absence of LMP expression which is invariably expressed during immortalization of normal B cells. This novel type of restricted gene expression could be compatible with evasion of host immune responses and consequent long-term survival of the cell in vivo.

Induction of latent membrane protein expression in in vitro Epstein-Barr virus-infected leukaemic B lymphocytes by interleukin 4 and antibodies to CD40.

Chronic lymphocytic leukaemia (CLL) B cells are clones representing the mature B cell phenotype. On infection with Epstein-Barr virus (EBV) CLL cells express the EB nuclear antigen (EBNA) complex but unlike EBV-infected normal B cells they do not express LMP nor do they proliferate or immortalize. Furthermore, EBV-CLL rapidly die by apoptosis in culture. In the present study we have used the B cell growth factors interleukin 4 and antibodies to CD40 to induce activation and proliferation of EBV-infected CLL cells. Although cell numbers did not significantly increase, apoptosis was partially inhibited in CLL cells which expressed increased levels of CD23 and were activated to immunoglobulin-secreting lymphoblasts. Expression of LMP was induced by interleukin (IL)-4 and anti-CD40 in all five EBV-infected CLL samples examined. However, this did not enhance cell proliferation or induce immortalization. Further analysis showed that LMP could be detected 4-5 days after EBV infection, and that both IL-4 and anti-CD40 could independently induce LMP but that their effect was additive. These results indicate that LMP expression is dependent on B cell activation processes and that in some circumstances full latent viral gene expression is not sufficient to cause B cell immortalization.

Epstein-Barr virus infection of HIV-seropositive individuals is transiently suppressed by high-dose acyclovir treatment.

OBJECTIVE: To assess whether oral acyclovir can eliminate persistent Epstein-Barr virus (EBV) infection and thereby prevent EBV-associated lymphoma development in HIV-seropositive homosexual men. METHOD: Persistent EBV infection was examined in a group of 21 HIV-seropositive homosexual men before, during and after treatment with oral acyclovir at a dose of 800 mg every 6 h (10 individuals) or with a placebo (11 individuals). RESULTS: In 13 individuals, EBV was isolated from the oropharynx before and after treatment (seven cases from the acyclovir-treated group and six from the placebo-treated group). A significant reduction in virus isolation occurred during treatment in the acyclovir-treated group, but not in the placebo-treated group. In seven cases in whom EBV shedding was detected before and after treatment, the EBV strain isolated was identical throughout the study, even when acyclovir had abolished detectable shedding for the duration of the treatment. In two other cases more than one strain was detected. On examination of the EBV type present, 89% of a group of 18 patients consistently shed type A virus, 5.5% type B virus and 5.5% showed evidence of co-infection with both virus types. This compares with figures of 100, 0 and 0%, respectively, in a control group of HIV-seronegative individuals. CONCLUSIONS: High-dose acyclovir therapy does not eliminate persistent EBV infection from the oropharynx of healthy HIV-seropositive individuals and therefore would not necessarily prevent lymphoma development. Our results suggest that infection by type B EBV, and co-infections of both A and B type, are more common in HIV-seropositives than HIV-seronegatives.

Epstein-Barr virus infection and immunity in bone marrow transplant recipients.

Studies on patients for up to one year following allogeneic, HLA-matched bone marrow transplants have shown no increased incidence of salivary Epstein-Barr (EB) virus secretion and no significant rise in EB-virus-specific antibody titers. EB-virus-specific cytotoxic T cells could be detected in the peripheral blood of all patients by six months posttransplant. For up to one year posttransplantation in vitro EB virus infection of peripheral blood B lymphocytes from the majority of patients leads to an abortive infection followed by cell death, and without the establishment of continuously growing cell lines. This abnormality appeared to be due to patients' monocytes, which formed a defective feeder cell layer in culture, and it could be circumvented by culturing the EB-virus-infected B cells from patients on a feeder layer of x-irradiated adherent cells from normal peripheral blood. These findings may explain the relative lack of EB-virus-associated lymphoma seen in bone marrow transplant recipients when compared with other groups of transplant patients.

Epstein-Barr virus associated Burkitt lymphoma in a heart transplant recipient.

This report describes a case of Epstein-Barr [correction of Epstein Barr] Virus related post-transplantation B lymphoproliferative disease of unusual Burkitt lymphoma (BL) type in which combined cytotoxic agents (PACE BOM) successfully produced complete and long-term tumor remission.

A prospective study in heart and lung transplant recipients correlating persistent Epstein-Barr virus infection with clinical events.

BACKGROUND: A 2-year prospective study was set up with 30 cardiothoracic transplant recipients to study Epstein-Barr virus (EBV) infection and immunity and their correlation with clinical events. METHODS: Regression assays were used to measure EBV-specific cytotoxic T lymphocyte (CTL) function. Tissue culture, immunoblotting, and polymerase chain reaction were used for EBV detection and isolate variation studies. RESULTS: CTL activity was significantly lower in pretransplant seropositive patients than in healthy controls (P<0.001). CTL response was undetectable in all patients during the first 6 months after transplantation, but returned at levels significantly lower than pretransplant and control levels during the second posttransplant year (P<0.001). Return of CTL function was directly correlated with time of last treated rejection episode (P<0.003) and duration of high plasma levels of cyclosporine (over 400 ng/ml; P<0.003). Significantly higher levels of EBV were detected in peripheral blood during the first 6 months than in pretransplant or control samples (P<0.05). Excretion of EBV in throat washings was significantly lower during the first 3 months when all patients were receiving acyclovir than in pretransplant and control samples (P=0.02). An increase in virus shedding was noted 3-6 months after transplantation, which was significantly higher than in pretransplant patients and controls (P<0.05). Comparison of recipients' and donors' virus isolates in 11 cases showed that seropositive recipients retained their original EBV isolate and did not acquire the donor virus. CONCLUSIONS: Immunosuppression decreased EBV-specific host immune function, which in turn favored increased EBV load in peripheral blood and increased excretion in the oropharynx. The transfer of donor virus to the seropositive recipients was not observed.

Immunological characterization of hemopoietic cells in the common marmoset, rhesus monkey, and man. In search of a model for human marrow transplantation.

Bone marrow, lymphoid, and peripheral blood cells from the common marmoset and rhesus monkey have been tested with a panel of heterologous and monoclonal antibodies, and their reactivity pattern has been compared with that of blood and bone marrow cells from human donors. Conventional antibodies reveal extensive cross-reactivity within the B cell, T cell, and granulocytic systems in all three species, however, some important differences have been exposed. Only the monoclonal antibodies to HLA-A,B,C and Ia-like antigens react with marmoset cells, and we have exploited this finding to show that the vast majority of colony-forming units (CFU-c) in the marmoset bone marrow (as in man) are Ia positive. The use of the common marmoset as a suitable model for human bone marrow transplantation is discussed in the light of these findings.

Risk of lymphoid neoplasia after cardiothoracic transplantation. a cohort study of the relation to Epstein-Barr virus.

BACKGROUND: Organ transplantation is associated with a greatly increased risk of Epstein-Barr virus (EBV)-associated lymphoproliferative disease (LPD), which is often fatal. There has been little epidemiological analysis, however, of the risk factors for LPD in transplant patients and none on whether the risks of non-EBV-associated lymphoid neoplasms are also increased. METHODS: The risk of lymphoid neoplasia was assessed in a cohort of 1563 patients who underwent cardiothoracic transplantation at Harefield Hospital, UK from 1980 to 1994 and were followed until December 1995. EBV antibody was assessed in the patients before transplantation, and lymphoid neoplasms were assessed for EBV RNA and latent EBV gene expression. RESULTS: Thirty cases of LPD occurred during follow-up. One lymphoma of unknown EBV status occurred. There were also six cases of EBV-negative non-Hodgkin's lymphoma (EBV-negative NHL), a highly significant excess over expectations from the general population rates of NHL (standardized incidence ratio 10.2 [95% confidence interval, 4.6-22.8]). The risk of LPD was significantly 10-fold raised in individuals who were EBV seronegative before transplantation; independently of this, it decreased steeply with age at transplantation and was greatest in the first year after transplantation. The risk was significantly raised in young seronegative recipients if the donor was older than the recipient. EBV-negative NHL occurred entirely in men 45 years old and older who were EBV seropositive before transplantation, and risk was not related to duration since transplantation. CONCLUSIONS: The risk factors found for LPD accord with EBV etiology and with greater hazard from primary infection than from reactivation. A second non-Hodgkin's lymphoid neoplasm, not related to EBV, seems also to be a consequence of transplantation and immunosuppression but is unlikely to be due to first infection by a ubiquitous agent. Its etiology and prevention need investigation separately from LPD.

Complete regression of posttransplant lymphoproliferative disease using partially HLA-matched Epstein Barr virus-specific cytotoxic T cells.

BACKGROUND: Adoptive immunotherapy with autologous and donor-derived cytotoxic T lymphocytes (CTL) has recently been used to treat Epstein Barr virus (EBV)-positive posttransplant lymphoproliferative disease (PTLD). METHODS AND RESULTS: We report complete regression of EBV-positive PTLD in an 18-month-old small bowel and liver transplant recipient after one infusion of partially human leukocyte antigen (HLA)-matched EBV-specific CTL grown ex vivo from an EBV seropositive unrelated blood donor. No infusion-related toxicity or evidence of graft-versus-host disease was observed. The tumor showed signs of regression within 1 week and EBV load in peripheral blood dropped to undetectable levels. Limiting dilution analyses (LDA) detected no EBV-specific CTL precursor (CTLp) cells before the infusion, and high numbers of CTLp at 4 hr and 24 hr post-CTL infusion. There was a reversal of the CD4/8 ratio in peripheral blood and an increase in HLA-DR positive CD8 cells. The patient has been in complete remission for 24 months. CONCLUSION: If this success is repeated in more PTLD patients, then stored CTL could be used for antiviral and antitumor therapies in immunocompromised patients.

Epstein-Barr virus (EBV) associated B-cell lymphoproliferative disease following HLA identical sibling marrow transplantation for aplastic anaemia in a patient with an EBV seronegative donor.

BACKGROUND: B-cell lymphoproliferative disorders (BLPD*) caused by Epstein-Barr virus (EBV) occurring after allogeneic bone marrow transplantation (BMT) are usually of donor origin. Treatment such as discontinuation of immunosuppression may be successful in some cases, but infusion of donor T cells results in successful eradication of EBV BLPD in most cases. METHODS AND RESULTS: We report a case of EBV positive aggressive BLPD after HLA matched sibling BMT for aplastic anaemia. The tumour completely regressed after withdrawal of cyclosporin and donor lymphocyte infusion. However, although the tumor was of donor origin, the donor serum was negative for antibodies to EBV antigens and no EBV-specific cytotoxicity was detected in donor peripheral blood mononuclear cells. The recipient was seropositive for EBV before BMT. CONCLUSIONS: We speculate that a 'second primary' EBV infection occurred involving donor cells in the recipient during BMT immunosuppression, with subsequent outgrowth of donor-derived BLPD. EBV infection may have been by an endogenous EBV isolate, from external sources, or from third party transfusions.

Immunohistology of Epstein-Barr virus-associated antigens in B cell disorders from immunocompromised individuals.

Proliferating B cell lesions developing in a series of immunosuppressed organ transplant recipients and patients with X-linked lymphoproliferative syndrome were examined for Epstein-Barr virus and cellular gene expression using immunocytochemistry and immunoblotting techniques. Results indicate that all the lesions examined from the patients in this series expressed Epstein-Barr virus gene products that were consistent with a latent, nonproductive type of infection. No lytic cycle antigens associated with productive viral infection were detected. This pattern is similar to the viral gene expression in normal B cells immortalized by Epstein-Barr virus in vitro. The demonstration in this study of Epstein-Barr virus viral gene expression in posttransplant and X-linked proliferative syndrome B cell disorders provides important new evidence for the primary role of Epstein-Barr virus in the development of these lesions. This is in contrast to the subsidiary role that the Epstein-Barr virus has in the etiology of Burkitt's lymphoma.

Current concepts in rational therapy for haemochromatosis.

Genetic haemochromatosis is characterised by an inappropriately high rate of iron absorption by the small intestine. The disease is transmitted as an autosomal recessive condition. The gene frequency in the Caucasian population is approximately 1 in 20 and the disease frequency is 1 in 400. Excessive iron deposition occurs in the liver, pancreas, heart, pituitary and joints and hepatic iron concentrations above approximately 400 mumol/g dry weight are always associated with fibrosis and usually with cirrhosis and progressive liver failure. Accurate diagnosis depends upon the demonstration of elevated hepatic iron stores. An hepatic iron index [hepatic iron concentration (in mumol/g dry weight) divided by patient age] of greater than 2.0 distinguishes homozygous subjects from the other conditions in which slight increases in hepatic iron concentration may occur, e.g. in a subject heterozygous for haemochromatosis or alcoholic liver disease. If cirrhosis is present, patients are at a high risk of developing hepatocellular carcinoma. Therefore, they should undergo regular abdominal ultrasound and alpha-fetoprotein estimation. In the absence of cirrhosis, phlebotomy restores life expectancy to normal. Venesection should be continued until all excess iron stores are removed as judged by failure of a rise in haemoglobin concentration on cessation of phlebotomy. Screening of first degree relatives should commence from a young age (e.g. 10 years). If serum ferritin or transferrin saturation are abnormal, liver biopsy should be undertaken. HLA typing of the family allows for the identification of those siblings who are most likely to develop the disease. Secondary iron overload is often multifactorial in origin. Iron chelation therapy with subcutaneous deferoxamine (desferrioxamine) should only commence after careful consideration of the potential benefits in each individual patient.