key: cord-0005574-c7wk9pnf authors: Blijlevens, NMA; Donnelly, JP; De Pauw, BE title: Mucosal barrier injury: biology, pathology, clinical counterparts and consequences of intensive treatment for haematological malignancy: an overview date: 2000-06-19 journal: Bone Marrow Transplant DOI: 10.1038/sj.bmt.1702447 sha: 09a1199fdaef1f2e2742272ac3708df4f4dff0eb doc_id: 5574 cord_uid: c7wk9pnf Mucositis is an inevitable side-effect of the conditioning regimens used for haematopoietic stem cell transplantation. The condition is better referred to as mucosal barrier injury (MBI) since it is primarily the result of toxicity and is a complex and dynamic pathobiological process manifested not only in the mouth but also throughout the entire digestive tract. A model has been proposed for oral MBI and consists of four phases, namely inflammatory, epithelial, ulcerative and healing phases. A variety of factors are involved in causing and modulating MBI including the nature of the conditioning regimen, the elaboration of pro-inflammatory and other cytokines, translocation of the resident microflora and their products, for example, endotoxins across the mucosal barrier, exposure to antimicrobial agents and whether or not the haematopoietic stem cell graft is from a donor. Neutropenic typhlitis is the most severe gastrointestinal manifestation of MBI, but it also influences the occurrence of other major transplant-related complications including acute GVHD, veno-occlusive disease and systemic infections. The pathobiology, clinical counterparts and the means of measuring MBI are discussed together with potential approaches for prevention, amelioration and, perhaps, even cure. Bone Marrow Transplantation (2000) 25, 1269–1278. Oral MBI is reported to affect 60% to 100% of transplant recipients 2,3 and is characterised by pain, oedema, erythema, lesions, pseudomembrane formation, excessive mucous production, reduced saliva and bleeding, all of which reduce the patient's ability to eat and drink. In contrast, there are no reliable data on the incidence of gut MBI although intestinal symptoms affect almost every transplant recipient to some extent and include nausea, vomiting, abdominal cramping and watery diarrhoea occasionally accompanied by macroscopic blood loss. The exact course and severity of bowel symptoms of MBI are also difficult to ascertain because many patients are in such pain due to oral MBI that they only gain relief from narcotic analgesia which induces constipation as a result of reduced gut motility. There are also a number of scoring systems for oral MBI 4 although none is universally accepted and all lack standardisation. As yet, there is no system for registering gut MBI although there are published definitions for grading toxicity of individual signs and symptoms. Consequently, much more is known about the course of oral MBI than its intestinal counterpart. Oral MBI is known to begin around the time conditioning therapy is completed, and has been shown to worsen until a peak is reached after which it declines gradually until resolving completely. The onset and duration of mucositis has also been shown to mirror the course of neutropenia 5 (Figure 1 ). This phenomenon may not be peculiar to any one specific regimen. It would therefore be of considerable interest were gut MBI shown to follow a similar course to oral MBI. MBI is a complex process that diminishes the quality of life and can predispose to more serious clinical complications including disseminated infection, veno-occlusive disease, acute graft-versus-host disease (GVHD) and even death. However, it seems more likely that gut MBI rather than oral MBI will present a greater risk to the patient even though it goes largely unrecognised. A pathobiological model for oral mucositis has recently been proposed which attempts to incorporate and explain all that is currently known about oral MBI. 6 This model describes four successive phases: (1) an inflammatory phase followed by (2) an epithelial phase leading to (3) an ulcerative/bacteriological phase and ultimately resolving in (4) the healing phase. This model could also be applicable to the gut as a whole even though it is a more complex organ having a dynamic epithelial border with different functions and unique interactions with immune system and luminal microflora 7 (Figure 2 ). The relationship between oral mucositis and neutrophil counts of allogeneic haematopoietic stem cell transplant recipients. Twenty-eight patients received idarubicin, cyclophosphamide and TBI 9 Gy as conditioning therapy for an allogeneic haematopoietic stem cell transplant. The course of mucositis closely mirrors that of neutropenia. Donnelly et al 5 1992. Radiation and cytotoxic drugs induce the systemic release of pro-inflammatory cytokines, interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF-␣) from activated macrophages and monocytes. Ionising radiation also induces cytokine gene expression directly. 8 TNF-␣ and its receptors are activated and suggest that resident tissue macrophages and monocytes rather than circulating polymorphonuclear (PMN) cells are the main target in vivo. 9 Tissue macrophages are not eliminated by conditioning therapy and may persist up to 4 months after transplantation. In the gut, macrophages reside in the gastrointestinal-associated lymphoid tissue (GALT) which houses the vast majority of total circulating lymphocytes as well as other members of the lymphoreticular system such as monocytes and intraepithelial lymphocytes. Once released into the circulation, the cytokines increase expression of HLA histocompatibility antigens and critical adhesion molecules that amplify local tissue injury by inviting PMN cells and activated lymphocytes to invade. 10 This results in increased vascularity and probably higher local levels of cytotoxic agents. In an animal model, exposure to bleomycin or 5fluorouracil (5-FU) resulted in increased cellularity of subepithelial oral tissue, vascular dilation and leukocyte margination within 24 h. 11 The generation of cytokines is self-limited during autologous transplantation and resolves within 7-10 days. 10 The inflammatory response may be specific to different classes of chemotherapeutic agents and to the particular Mucosal barrier injury. Mucosal barrier injury occurs in four phases. The first phase is the inflammatory/vascular phase and is characterised by the induction of pro-inflammatory cytokines IL-1, TNF-alpha and IFN-gamma by cytotoxic drugs and irradiation while the epithelial cells are still intact. The second phase is the epithelial phase when cells cease dividing and die. This coincides with neutropenia. The third phase is when necrosis and ulceration occur and is when the resident microbial flora and their products, eg endotoxin translocate into the bloodstream. Moreover, impaired local defences and lower levels of secretory IgA may allow local infection to develop. The final phase is when healing takes place and involves the action of naturally occurring substances including trefoils, EGF and TGF. The events that take place in the gut are almost certainly more complicated than those occurring in the oral cavity since the gastrointestinal tract is intrinsically more complex in terms of its function, it possesses the specialised gastrointestinal-associated lymphoid tissue (GALT) system, and its resident microflora are more numerous and varied. sequence of preparative regimens used in haematopoietic stem cell transplantation since a variety of different profiles of cytokine release have been reported. [12] [13] [14] For instance, elevated TNF-␣ levels were found in 13 (24%) of 56 patients given either cyclophosphamide and total body irradiation or cyclophosphamide and busulphan and these levels were predictive for transplant-related complications within the first 6 months post BMT. 14 Serum levels of TNF-␣ and IL-1␤ have also been shown to be markedly higher with higher doses of TBI 1 week after transplant. 13 In contrast, busulphan, VP-16 and cyclophosphamide induced interferon-gamma production directly. 12, 15 Moreover, intestinal damage manifest by villous blunting, apoptosis and brush border loss (the surface area of villous cells amplified by numerous finger-like microvilli) correlated well with cytokine levels. 13 In a clinical phase I/II study, use of the monoclonal antibody MAK 195F diminished the release of TNF-␣ but it was also observed that the kinetics of TNF-anti-TNF complexes were different after conditioning therapy with cyclophosphamide and TBI compared with cyclophosphamide and busulphan, which actually induced less TNF-␣ release. 16 Elevated cytokine levels detected as early as 1 week after transplantation might be related to engraftment in the absence of complications or to infectious disease, non-infectious events or GVHD rather than MBI. 17 Moreover, other investigators have failed to find elevated levels of cytokines during or shortly after conditioning therapy. 12, 17, 18 Before total cell destruction, TNF-␣, IFN-␥ and IL-1 induce major changes in the functionality, permeability, brush border transport, glutamine utilisation (glutamine is the main source of energy for intestinal cells) and mucosal cell integrity. [19] [20] [21] [22] IFN-␥ and TNF-␣ induce dose-related cellular exfoliation, leading to the formation of a mucoid cap in a vain attempt to protect the mucosa. 23 Epithelial cells are also capable of producing and secreting TNF-␣ and IL-1␣. 24 In an H-2-incompatible transplanted SCID mice model, colonic TNF-␣, IL-1␣ and IL-6 appeared 4 h after TBI and peaked by 24 h. If no transplantation followed, TNF-␣ and IL-1␣ levels decreased rapidly 3-5 days later. 17, 18, 25 Epithelial cells are also capable of mounting an immune host response, and of taking up, processing and presenting soluble antigens as well as expressing MHC class II molecules. 26 Thus, taken together, these data support the view that the primary step in MBI is an inflammatory response. Cytotoxic drugs and radiation interfere with rapidly dividing cells and the kinetics of proliferating mucosal cells influence their sensitivity to these agents. Normally, cell renewal takes place continuously in crypts from a proliferating pool of clonal undifferentiated stem cells and cell division is completed in about 24 h. 27 Younger cells migrate up to the villous tips and slough off into the lumen at the extrusion zone. Anti-metabolites, for example cytarabine, are cell cycle-dependent and interfere with the synthesis of DNA in dividing cells whereas the intercalating agents such as the anthracyclines are more effective during the G2 phase after mitosis is complete when the cell has time to restore errors. In contrast, alkylating agents such as cyclophosphamide generate lethal DNA lesions even in resting cells by forming cross-links between DNA strands while ionising radiation exerts its main effect during mitosis. Various chemotherapeutic drugs such as adriamycin, bleomycin and 5-FU increase cellular sensitivity to radiation in a synergistic manner. 28 This has also been observed clinically when idarubicin was given at the same time as cyclophosphamide and TBI. 29 Normally, the entire epithelium is renewed in 4-6 days, but decreased cell renewal is thought to lead to mucosal atrophy, thinning and necrosis although in rats, sublethal doses of alkylating agents mainly induced lower absorption rather than villous atrophy. 30 The ulcerative-bacteriological phase Increased redness and swelling of the mucosa and underlying tissue are usually the first signs of oral MBI, mainly due to increased vascularity and vascular permeability (inflammatory phase) and thinning of epithelium (epithelial phase). This process usually culminates in the ulcerative phase within about 14 days of starting chemotherapy. 1 It is also during this phase that the resident microflora are Bone Marrow Transplantation assumed to play a role. Normally, these microorganisms contribute to maintaining the integrity of the integument and prevent pathogenic microbes from gaining a foothold. The ecological system tries to maintain its balance but once the mucosa is damaged, microbes may infect the submucosal tissue. Non-pathogenic streptococci specifically bind to, and use, the glycoproteins in the dental plaque that develops in the absence of normal food intake and saliva production. The non-cellular defence depends on amount and quality of mucus and saliva produced which contain diverse host defence peptides (defensins), lactoferrin, lysozyme and immunoglobulins. 31 Secretory IgA inhibits bacterial adherence, specifically of oral streptococci, neutralises toxin and virus, prevents antigen uptake and possesses anti-inflammatory activity. 32 Several classes of host defence peptides can be found in saliva and on surfaces each possessing rapid lytic activity against the membranes of Grampositive and Gram-negative bacteria as well as yeasts. 33 During conditioning therapy and after transplantation, the salivary immunoglobulins (IgA, IgG, IgM) have been shown to be lower than normal and the elimination of T cells from engrafted bone marrow results in less initial capacity for immunoglobulin production and secretion. 34 Thus, the local immune defences of the oral cavity are impaired. It is common practice to administer antimicrobial agents, particularly the fluoroquinolones and local antiseptics such as chlorhexidine, to haematopoietic stem cell transplant recipients, leading inevitably to marked shifts in the resident oral flora towards the more resistant species particularly the viridans (alpha-haemolytic) streptococci. This shift is more profound in patients with overt oral mucositis. 35 There has also been a corresponding increase in bacteraemia due to these streptococci with oral mucositis being an important risk factor in autologous haematopoietic stem cell transplant recipients. 36 Similarly, Donnelly et al 37 reported a higher incidence of viridans streptococcal bacteraemia due to the marked mucositis associated with treatment intensification. One particular species, Streptococcus mitis, is apparently associated with sepsis and adult respiratory distress syndrome (ARDS), mainly after high-dose cytarabine. 38, 39 This syndrome could be provoked by changes in the pulmonary endothelium and lung macrophages induced by cytotoxic chemotherapy which, in turn, induces cytokine production perhaps triggered by infection with Streptococcus mitis. 40 The stomach or small intestine could also be a portal of entry if colonisation with these streptococci occurs as a result of the achlorhydria induced by H 2 histamine antagonists and proton pump inhibitors since the use of these agents has been noted as a risk factor for the so-called 'alpha-strep syndrome'. 38 Obviously, MBI is itself a risk factor for viridans streptococcal bacteraemia but it might not always indicate systemic infection since transient bacteraemia also occurs in healthy persons after dental manipulation. 41 Moreover, these bacteria do not elaborate exotoxins nor are they professional pathogens. Thus, viridans streptococcal bacteraemia might simply signal the presence of mucosal barrier injury rather than infection. Although, to some extent, similar to the oral cavity, the gut harbours a much more complex ecosystem comprising a greater variety of aerobic and anaerobic bacteria that share a symbiotic relationship with the host. This relationship plays an important role in maintaining the gut's histological structure and also provides so-called 'colonisation resistance', ie the ability of the gut to repel foreign bacteria. The intestinal microflora depends on prebiotics, the fibrous nutrients 42 that enhance probiotic bacteria, like bifidobacteria, lactobacilli and Clostridium species. These are the species that are thought to provide the colonisation resistance by elaborating antibacterial compounds and competing for nutrients so preventing overgrowth by potentially pathogenic bacteria. 43 These probiotic bacteria also produce nutrients for mucosal cells. Certain antimicrobial agents, particularly those that affect cell wall synthesis, exert a major impact on the gut's ecosystem by destroying the 'protective' anaerobic flora particularly the probiotic bacteria. When the gut epithelium is disrupted, bacterial translocation occurs and pro-inflammatory bacterial oligopeptides, especially endotoxin (lipopolysaccharide or LPS) readily gain access. 44 In the normal host (whether animal or human) pathogenic bacteria such as Escherichia coli and Pseudomonas aeruginosa penetrate the mucosa and migrate to extra-intestinal sites such as the mesenteric lymph nodes, spleen and liver. The GALT system, together with the Kupffer cells of liver and spleen serve as a backup to trap endotoxins and kill bacteria. The rate of translocation of enterobacteria like E. coli and other gram-negative bacilli such as Pseudomonas aeruginosa is strongly associated with the degree of neutropenia. 45 Microbial translocation is exacerbated by irradiation 46 and chemotherapy 47 as microorganisms can be cultured in extra-intestinal sites as well as in blood. 48 Different modes of translocation exist and occur even before any histological damage is apparent. Anaerobic non-pathogenic bacteria rarely translocate but yeasts such as Candida albicans can do so more easily when disruption has occurred. 49 Endotoxin can be transported through the lymphatic channels, bypass the liver or enter the peritoneal cavity directly and can cause systemic endotoxaemia. 50 Endotoxin can also increase intestinal permeability directly 51 or by stimulating primed macrophages to release an excessive amount of cytokines, mostly TNF-␣, thereby inducing mucosal inflammation and increasing permeability. 52 Higher levels of circulating endotoxin are obtained after giving intensive TBI containing regimens 13 suggesting that persistent low-grade endotoxaemia or the inflammation associated with MBI induce fever of unknown origin since endotoxaemia and gut mucosal damage occurred in 44 (70%) of 63 HSC transplant recipients (both allogeneic and autologous) all of whom developed fever that could not be explained by infection. 53 Peptidoglycan (the major component of the cell wall of Gram-positive bacteria) may play a similar role as endotoxin as it is also biologically active in tissues and may induce a pro-inflammatory response. 54 Although much less potent than endotoxin gram-for-gram, large amounts of peptidoglycan may well be released into the circulation when gut MBI is present simply because there are many more Gram-positive than Gram-negative bacteria in the gut. Exposure to antibiotics that cause lysis will also liberate cell wall fragments. Typhlitis, also called neutropenic enterocolitis, necrotising enterocolitis or ileocaecal syndrome, is a caecitis often extending to both the proximal and distal caecum that may be primarily a severe manifestation of gut MBI. Indeed, all factors that contribute to the development of MBI are present clinically. First, typhlitis occurs after the administration of cytotoxic drugs, particularly high-dose cytarabine, etoposide and anthracyclines at the nadir of neutropenia and thrombocytopenia. Secondly, prolonged exposure to antibiotics results in a marked shift in the gut microflora towards toxin producing bacteria 55 such as Staphylococcus aureus, Pseudomonas aeruginosa and Clostridium septicum. 56, 57 In fact, Clostridium species are now more likely to predominate for reasons which are poorly understood so that bacteraemia due to C. tertium or C. septicum is almost pathognomonic for typhlitis. Antimicrobial pressure also predisposes to intestinal overgrowth by Clostridium difficile in transplant recipients. 58 Necrosis of the mucosal surface of the ileocaecal region probably provides a favourable environment for the spores of Clostridium species to germinate and may be their portal of entry into the bloodstream. The pathogenesis of typhlitis would therefore seem to require various elements to be present simultaneously, namely gut MBI, a perturbed resident microflora and profound neutropenia. Typhlitis is not only a paradigm for MBI but, because of the high mortality rate, it is also the most severe clinical form of MBI and deserves more attention both in terms of developing techniques for early diagnosis as well as in evolving strategies for prevention and treatment. Consequently, we can expect to encounter more cases of typhlitis as chemotherapeutic regimens become more intense. In general, the repair of oral MBI parallels haematological reconstitution as peripheral blood counts return to normal 3,5,59 with complete resolution occurring within 2-3 weeks. 60 In contrast, gut function does not return to normal for several more weeks, since malabsorption and diminished enzyme activity still persist even after structural repair. The healing of mucosal damage probably occurs in two phases commencing with the restitution of mucosal integrity and then remodelling of the mucosal architecture. The mucosal repair process depends on the severity of damage since superficial injury can be repaired rapidly by epithelial cell migration without mitosis. 61 However, proliferation in conjunction with angiogenesis is necessary for deep lesions, involving large areas of necrosis, to recover. 62 Trefoil peptides (mucin-associated peptides) are secreted by epithelial cells, with each region of the gut probably having its own variant. 62 These peptides act as rapid response molecules to injury by promoting cell migration, cell differentiation and wound healing. 63 Epidermal growth factor, transforming growth factor alpha, interleukin-11 and fibroblast growth factor also appear to promote epithelium repair and regeneration. 64 In contrast, these agents appear to play only a limited role in the healing of the oral mucosa. Gut MBI may evolve into acute GVHD since tissue damage caused by conditioning regimens plays a role in both conditions. Any mature donor T lymphocytes within the allograft that recognise host-antigens are activated by endotoxin and pro-inflammatory cytokines. 10, 13 Animal studies 25 and some studies in humans 13, 14 suggest that high levels of pro-inflammatory cytokines predispose to acute GVHD, although others could not confirm this observation. 12, 15, 17, 65 The early administration of the anti-TNF␣ monoclonal antibody MAK 195F changes the nature of the inflammatory response, reduces the number of febrile episodes and delays the onset and severity of acute GVHD. 16 The microflora might also play a role in triggering acute GVHD because less disease was observed in decontaminated murine chimeras. 66, 67 Intestinal decontamination with metronidazole also significantly reduced the severity of acute GVHD in HLA-identical sibling transplants. 68 Taken together, these data suggest a role for MBI in triggering acute GVHD because of either the release of cytokines induced by conditioning regimens or the translocation of microbial toxins. Permeability refers to the property possessed by a epithelium that enables passage of a solute by unmediated diffusion. 69 Permeability can be measured in vivo by means of the urinary excretion of test substances or by detecting their presence in blood. Lactulose, various polymers of polyethylene glycol (PEG) or 51 Cr-labelled ethylenediaminetetraacetic acid ( 51 Cr-EDTA) have all been used, but the results are markedly influenced by extraneous factors such as bowel transit time, gastric emptying and renal function. Nonetheless, permeability to 51 Cr-EDTA is increased as soon as 2 days after starting conditioning therapy and continues to increase until shortly after BMT, about 12 days later. 70 Others have shown that the intestinal toxicity induced by melphalan can be monitored using 51 Cr-EDTA thus allowing the effectiveness of various treatments for reducing the intestinal toxicity to be assessed. 71 Unfortunately, 51 Cr-EDTA is radioactive and not suitable for routine use. The uptake of antibiotics such as gentamicin and tobramycin may provide a safer means of determining increased permeability since such drugs are normally excluded by the intact gut but can be detected in plasma during mucositis when given by mouth. 72 Studies of epithelial cell handling of cytotoxic drugs, radiation and antimicrobial agents could offer new possibilities for documenting MBI. 73, 74 The principal features of gut MBI are a loss of epithelial surface and a change in the permeability. 69 This can also be measured if at least two different probes are used at once since the extraneous factors equally affect the pre-and postmucosal determinants and the urinary excretion ratio Bone Marrow Transplantation becomes an index of intestinal permeability. For example, monosaccharides such as mannitol and rhamnose are absorbed through aqueous pores in the cell membrane whilst disaccharides like lactulose gain access through the tight junctions located at the upper end of adjacent epithelial cells. Tight junctions are dynamic structures exerting physiologic control over the flow of solutes through paracellular spaces and play an important role in gut permeability. Reduction of urinary monosaccharide excretion represents a loss of epithelial cell surface area, while increased urinary disaccharide excretion indicates damage to the tight junctions. Sugar absorption tests (SAT) have proved their value in intestinal diseases but they lack diagnostic specificity. 75 SATs offer an easy, reliable means of assessing the onset, duration and severity of gut MBI in patients treated with cytotoxic agents. Absorption is increased after only 2 days treatment with chemotherapy 76 suggesting that cytokines might interfere with the tight junctions (see inflammatory phase) rather than directly inhibiting cell proliferation, which tends to occur later. 77 Altered permeability continues to progress until reaching a peak about 7 days after conditioning therapy has been completed 78 and returns to normal about 4 weeks later. 79 This mirrors the oral MBI and neutropenia (Figure 1) . It should be possible to discriminate patients at risk of developing serious toxicity to therapy from those not at risk by using these SATs since a positive correlation was found between progressive non-oral clinical toxicity and increased permeability in transplant recipients. 70, 71 Bow et al 80 also found that the absorption of D-xylose was at its lowest 2-3 weeks after remission induction treatment had been started in patients who developed systemic candidosis. Malabsorption of D-xylose was also found to be an independent predictor of neutropenic enterocolitis and hepatosplenic candidosis and also correlated well with bacteraemia. 80, 81 As yet, there are no objective means of determining gut MBI in use routinely and none has been validated for use in clinical trials to assess gut toxicity although the data available suggest SATs may be useful for helping adapt supportive care regimens for selected patients in order to reduce morbidity and perhaps mortality. A consensus in the way MBI is measured in clinical practice analogous to the validated scoring system of oral mucositis of the Mucositis Study Group 82 is a prerequisite for such studies. There is also a pressing need for much simpler tests of each phase of MBI. Data from chemotherapyinduced cytokine expression of epithelial cells in singlecell testing or cell lines, quantitation of cytokine profiles in saliva or stools 83 and the results of basal cell kinetic studies like grading of epithelial cell viability by trypan blue dye exclusion obtained by oral washings 84 should be incorporated in clinical research and care. It should be possible to demonstrate overt mucositis using radionuclide imaging techniques, such as indium-labelled leukocytes 85 and technetium-labelled diphytanoylphosphatidylcholine liposomes but there are only a few anecdotal reports and their clinical feasibility is expected to be minimal. Enteral nutrition stimulates gut-responsive hormones, prevents mucosal atrophy, improves mucosal blood flow and gastrointestinal motility, stimulates mucus formation and secretion of sIgA and reduces bacterial translocation. 86 In children without severe MBI, enteral and parenteral nutrition were equally effective in maintaining the nutritional status 87 and a diet containing lactose and bovine milk protein appeared to be well tolerated. 88 Oral administration of short-chain fatty acids (SCFAs) typically produced by the anaerobic flora of the gut reduces the inflammation and necrosis induced by cytarabine in mice. 89 These SCFAs are normally produced by the fermentation of dietary fibre and unabsorbed starch by the same gut microflora and are the preferred fuel of enterocytes. Total parenteral nutrition (TPN) is advocated for those patients who are either malnourished or who are expected to have inadequate oral intake for a prolonged period (usually 7-10 days) to restore the negative nitrogen and caloric balance. These patients are typically those with MBI that is sufficiently severe that it impedes adequate enteral nutrition leading to malnutrition, weight loss, malabsorption and micronutrient deficiencies. TPN does help to reduce the morbidity of malnourished patients completing a course of myeloablative therapy, 1 but at the same time it promotes villous atrophy, increases intestinal permeability, reduces luminal sIgA content and enhances bacterial translocation. 90 Nevertheless, the long-term outcome for allogeneic HSC transplant recipients is better with TPN, even when they are well-nourished 91 whereas autologous HSC transplant recipients gain little or no benefit. 92 Glutamine has attracted a lot of attention because it is the primary fuel for intestinal epithelia and the cornerstone of protein and nucleic acid synthesis but mucosal cells cannot synthesise enough themselves making glutamine conditionally essential during stress. 93 Administering glutamine to animals after irradiation and chemotherapy prevented mucosal atrophy and reduced bacterial translocation, endotoxaemia and infections. [94] [95] [96] It is much less clear whether glutamine given orally prevents human oral MBI, 97, 98 although patients treated with high-dose chemotherapy experienced less diarrhoea. 99 Glutamine supplementation given to HSC transplant recipients parenterally helps to preserve hepatic function, reduces the length of stay in hospital, improves the nitrogen balance and lowers the infection rate 100,101 but has no influence on the occurrence of mucositis or fever. 102, 103 However, nothing is known about the effect of glutamine on gut integrity or function since permeability tests were not performed. Direct cytoprotectants such as sucralfate and diphenhydramine do not ameliorate oral MBI 1,104-106 whereas indirect cytoprotectants, like transforming growth factor ␤3 and epi-dermal growth factor, interfere with epithelial cell replication in animals and are being tested in clinical trials for their efficacy and safety in modulating oral MBI. 107, 108 Recombinant-human GM-CSF given as a mouthwash shortened the duration of severe oral MBI 109, 110 but the mechanism of action remains unclear. GM-CSF might have a direct pleiotropic effect on epithelial cell kinetics. Alternatively, the effect may be indirect as a result of the first neutrophils produced by haematopoietic progenitor cells migrating to the oral mucosa and thereby reducing local infection. 111 Other clinical trials exploring the effects of recombinant growth factors such as transforming growth factor-␤1 or TGF-␤3 and others on MBI are coming. [112] [113] [114] A clearly different approach consists of delivering monoclonal antibodies that bind and inactivate doxorubicin (MAD11) in intestinal cells. 115 It is common practice to try to reduce the bioburden of gram-negative bacilli in the oral cavity by giving antimicrobial agents, and maintaining good oral hygiene and also to provide remedial dental treatment when necessary to reduce oral complications. 116 Antibiotic lozenges containing tobramycin, polymyxin and amphotericin B reduce oral MBI 117 but the effect of chlorhexidine is unclear. [118] [119] [120] [121] There have been no formal studies of the effect of antimicrobial agents whether given for prophylaxis or treatment on MBI although it is usually assumed that they are beneficial. If MBI is not primarily the result of infection as seems to be the case, treatment with antimicrobial agents is unlikely to be of benefit and may even prove harmful in exerting selective pressure on the resident flora. Probiotics may help restore the balance of gut flora in cancer patients 122 but trials of sufficient size are lacking. IgMenriched immunoglobulin has been shown to reduce endotoxaemia and febrile episodes in transplant recipients. 53 Mucosal barrier injury is far more than simply a toxicological side-effect of cytotoxic regimens. Enough evidence exists to indicate that MBI is a complex and dynamic pathological process but it is essential to understand its nature more fully. A model for oral MBI already exists and shows that it is the net result of an almost complete breakdown of the epithelium initiated by the release of pro-inflammatory cytokines induced by the cytotoxic drugs followed by an arrest of the mucosal cell cycle and inhibition of repair leading to apoptosis. Infection, if it plays any role at all, is largely secondary. This model may go some way to explain the corresponding phenomenona in the gut although gut MBI is likely to be much more complex and more difficult to unravel mainly because the damage cannot be seen and the signs and symptoms are too imprecise. Since gut permeability increases very soon after exposure to chemotherapy and irradiation it seems logical to pursue tests such as the SATs further and to look for other chemical probes. Certainly, a means of objectively monitoring MBI is necessary before drug products can be formally tested for their effects on MBI. At this moment there are several products ranging from cytokines and defensins to nutrients and probiotics which look promising. For example the growth factor interleukin-11 (IL-11) by reducing pro-inflammatory cytokine expression and secretion by macrophages (phase I), prevents apoptosis of intestinal crypt cells partially by inhibiting proliferation (phase II) and promotes recovery of these crypt cells while remodelling connective tissue (phase IV). Defensins, trefoil peptides and even sIgA-antibodies could offer additional tools to tackle hostile microbes, for example, IgA-IgG administered orally has been shown to reduce gut MBI in patients undergoing intensive cytotoxic therapy. 123 With the means of reliably detecting and monitoring gut MBI at our disposal, the process will graduate from being an expected although unpleasant side-effect with few therapeutic options to a condition that might actually be preventable. Cancer; Principles and Practice of Oncology Clinical assessment scale for the rating of oral mucosal changes associated with bone marrow transplantation. Development of an oral mucositis index A longitudinal study of oral ulcerative mucositis in bone marrow transplant recipients Scoring oral mucositis A scheme for daily monitoring of oral mucositis in allogeneic BMT recipients Mucositis as a biological process: a new hypothesis for the development of chemotherapy-induced stomatotoxicity Regulation of tumor necrosis factor gene expression by ionizing radiation in human myeloid leukemia cells and peripheral blood monocytes Evidence for induction of TNFa by irradiation and cytotoxic therapy preceding bone marrow transplantation -in vivo and in vitro studies Cytokine dysregulation as a mechanism of graft versus host disease An animal model for mucositis induced by cancer chemotherapy Serum levels of cytokines and secondary messages after T-cell-depleted and non-T-cell-depleted bone marrow transplantation: influence of conditioning and hematopoietic reconstitution Total body irradiation and acute graft-versus-host disease: the role of gastrointestinal damage and inflammatory cytokines Increased serum levels of tumor necrosis factor alpha precede major complications of bone marrow transplantation Analysis of serum levels of interferon and interferon-dependent secondary messages Modulation of acute graft-versus-host-disease after allogeneic bone marrow transplantation by tumor necrosis factor alpha (TNF alpha) release in the course of pretransplant conditioning: role of conditioning regimens and prophylactic application of a monoclonal antibody neutralizing human TNF alpha (MAK 195F) Serum cytokine levels in patients undergoing bone marrow transplantation Serum levels of interleukin 6, interleukin 8, and C-reactive protein after human allogeneic bone marrow transplantation IFN-gamma modulation of epithelial barrier function. Time course, reversibility, and site of cytokine binding Cytokine regulation of intestinal glutamine utilization Tumor necrosis factor-alpha increases sodium and chloride conductance across the tight junction of CACO-2 BBE, a human intestinal epithelial cell line Cytokine modulation of Na(+)-dependent glutamine transport across the brush border membrane of monolayers of human intestinal Caco-2 cells Cytokine effects in a human colonic goblet cell line. Cellular damage and its partial prevention by 5 aminosalicylic acid Cytokine mRNA expression in labial salivary glands and cytokine secretion in parotid saliva in Sjogren's syndrome Effect of total body irradiation, busulfan-cyclophosphamide, or cyclophosphamide conditioning on inflammatory cytokine release and development of acute and chronic graft-versus-host disease in H-2-incompatible transplanted SCID mice Regulation of the mucosal epithelial barrier Aspects of the biology of regeneration and repair in the human gastrointestinal tract Basic Clinical Radiobiology; for Radiation Oncologists Idarubicin-related side effects in recipients of T-cell-depleted allogeneic bone marrow transplants are schedule dependent Effects of cancer, radiotherapy and cytotoxic drugs on intestinal structure and function Host defense mechanisms at mucosal surfaces Intestinal IgA: novel views on its function in the defence of the largest mucosal surface Oral complications in bone marrow transplantation patients: recent advances Oral sources of septicaemia in patients with malignancies The impact of mucositis on alpha-hemolytic streptococcal infection in patients undergoing autologous bone marrow transplantation for hematologic malignancies Bacteremia due to oral viridans streptococci in neutropenic patients with cancer: cytostatics are a more important risk factor than antibacterial prophylaxis Septicemia and shock syndrome due to viridans streptococci: a case-control study of predisposing factors Changes in the oral streptococcal flora of children undergoing allogeneic bone marrow transplantation Pre-emptive administration of corticosteroids prevents the development of ARDS associated with Streptococcus mitis bacteremia following chemotherapy with high-dose cytarabine Bacteremia after oral surgery and antibiotic prophylaxis for endocarditis Probiotics, prebiotics or 'conbiotics'? Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics Bacterial chemotactic oligopeptides and the intestinal mucosal barrier Bacterial translocation and gram-negative bacteremia in patients with hematological malignancies Abdominal radiation causes bacterial translocation Bacterial translocation from the gastrointestinal tracts of mice receiving immunosuppressive chemotherapeutic agents Proposed mechanisms for the translocation of intestinal bacteria The process of microbial translocation Clinical significance of translocation A single dose of endotoxin increases intestinal permeability in healthy humans Macrophage priming and lipopolysaccharide-triggered release of tumor necrosis factor alpha during graft-versus-host disease Effect of IgMenriched intravenous immunoglobulin (Pentaglobin) on endotoxaemia and anti-endotoxin antibodies in bone marrow transplantation Bacterial peptidoglycan polysaccharides in sterile human spleen induce proinflammatory cytokine production by human blood cells Neutropenic enterocolitis: spectrum of the disease and comparison of definite and possible cases Editorial response: neutropenic enterocolitis Clostridium septicum sepsis and neutropenic enterocolitis in a patient treated with intensive chemotherapy for acute myeloid leukemia Clinical significance of alimentary tract microbes in bone marrow transplant recipients Stomatologic complications of bone marrow transplantation in a pediatric population Intestinal and hepatic complications of human bone marrow transplantation. Part II Epithelial restitution in the gastrointestinal tract Molecular aspects of mucosal repair: a summary (comment) Trefoil peptides: mitogens, motogens, or mirages? Healing the epithelium: solving the problem from two sides Serum cytokine levels in bone marrow transplantation: synergistic interaction of interleukin-6, interferon-gamma, and tumor necrosis factor-alpha in graft-versus-host disease The role of microflora in development of graft-versus-host disease Role of bacterial microflora in development of intestinal lesions from graft-versus-host reaction Influence of intestinal bacterial decontamination using metronidazole and ciprofloxacin or ciprofloxacin alone on the development of acute graft-versus-host disease after marrow transplantation in patients with hematologic malignancies: final results and long-term follow-up of an open-label prospective randomised trial Intestinal permeability: functional assessment and significance Gastro-intestinal toxicity related to bone marrow transplantation: disruption of the intestinal barrier precedes clinical findings 51Cr-EDTA test for intestinal permeability Absorption of oral aminoglycosides following bone marrow transplantation Epithelial cells in culture as a model for the intestinal transport of antimicrobial agents The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications Clinical implications of the sugar absorption test: intestinal permeability test to assess mucosal barrier function Changes in intestinal permeability to lactulose induced by cytotoxic chemotherapy Effects of anthracycline therapy on intestinal absorption in patients with advanced breast cancer Effect of highdose chemotherapy on intestinal permeability in humans The gut mucosal barrier in bone marrow transplantation Invasive fungal disease in adults undergoing remission-induction therapy for acute myeloid leukemia: the pathogenetic role of the antileukemic regimen Cytotoxic therapy-induced D-xylose malabsorption and invasive infection during remission-induction therapy for acute myeloid leukemia in adults Validation of a new scoring system for the assessment of clinical trial research of oral mucositis induced by radiation or chemotherapy Detection of proand anti-inflammatory cytokines in stools of patients with inflammatory bowel disease A new in vitro assay for quantitation of chemotherapy-induced mucositis Indium-labeled white blood cells apheresed from donors receiving G-CSF localize to sites of inflammation when infused into allogeneic bone marrow transplant recipients Bacterial translocation: the influence of dietary variables Nutritional support in children undergoing bone marrow transplantation Enteral nutrition after bone marrow transplantation Oral administration of short-chain fatty acids reduces the intestinal mucositis caused by treatment with Ara-C in mice fed commercial or elemental diets Total parenteral nutrition-induced changes in gut mucosal function: atrophy alone is not the issue Positive effect of prophylactic total parenteral nutrition on long-term outcome of bone marrow transplantation Parenteral nutrition following intensive cytotoxic therapy: an exploratory study on the need for parenteral nutrition after various treatment approaches for haematological malignancies Is glutamine a conditionally essential amino acid? Effect of glutamine on intestinal mucosal integrity and bacterial translocation after abdominal radiation Prophylactic glutamine protects the intestinal mucosa from radiation injury Glutamine facilitates chemotherapy while reducing toxicity Effect of lowdose oral glutamine on painful stomatitis during bone marrow transplantation Oral glutamine reduces the duration and severity of stomatitis after cytotoxic cancer chemotherapy Oral glutamine in the prevention of chemotherapy-induced gastrointestinal toxicity Parenteral glutamine protects hepatic function during bone marrow transplantation Clinical and metabolic efficacy of glutamine-supplemented parenteral nutrition after bone marrow transplantation. A randomized, doubleblind, controlled study Oral and parenteral glutamine in bone marrow transplantation: A randomized, double-blind study Parenteral glutamine dipeptide supplementation does not ameliorate chemotherapy-induced toxicity Effects of sucralfate on mucositis during and following radiotherapy of malignancies in the head and neck region. A double-blind placebo-controlled study Sucralfate mouth washing in the prevention of radiation-induced mucositis: a placebo-controlled double-blind randomized study The effects of sucralfate suspension and diphenhydramine syrup plus kaolinpectin on radiotherapy-induced mucositis Transforming growth factor-beta 3 mediated modulation of cell cycling and attenuation of 5-fluorouracil induced oral mucositis Effect of epidermal growth factor on ulcerative mucositis in hamsters that receive cancer chemotherapy Effect of granulocyte-macrophage colony-stimulating factor on oral mucositis after hematopoietic stem-cell transplantation Effect of granulocyte-macrophage colony-stimulating factor on chemotherapy-induced oral mucositis in non-neutropenic cancer patients Studies of oral neutrophil levels in patients receiving G-CSF after autologous marrow transplantation IL-11, a pleiotropic cytokine: exciting new effects of IL-11 on gastrointestinal mucosal biology Mitigating effects of interleukin 11 on consecutive courses of 5-fluorouracil-induced ulcerative mucositis in hamsters Preclinical and early clinical development of keratinocyte growth factor, an epithelial-specific tissue growth factor Oral delivery of antibodies. Future pharmacokinetic trends Current practices in the oral management of the patient undergoing chemotherapy or bone marrow transplantation Local antimicrobial therapy of oral mucositis in paediatric patients undergoing bone marrow transplantation Chlorhexidine for prophylaxis against oral infections and associated complications in patients receiving bone marrow transplants Randomized trial of a chlorhexidine mouthwash for alleviation of radiationinduced mucositis Oropharyngeal mucositis complicating bone marrow transplantation: prognostic factors and the effect of chlorhexidine mouth rinse Mucositis prevention by selective elimination of oral flora in irradiated head and neck cancer patients Clinical uses of probiotics for stabilizing the gut mucosal barrier: successful strains and future challenges Gut mucosa barrier preservation by orally administered IgA-IgG to patients undergoing bone marrow transplantation: a randomised pilot study