Berthou

MD; Adrian Tempescul
email: adrian.tempescul@chu-brest.fr

Jean-Christophe C Ianotto

Jean-Richard R Eveillard

Gaelle Guillerm

Christian Berthou

Dr Tempescul Adrian

ESHAP chemotherapy regimen associated to Lenalidomide induce complete isotopic remission in Hodgkin's lymphoma relapsing after autologous stem cell transplantation

Keywords: hodgkin lymphoma relapse autograft chemotherapy lenalidomide Hodgkin lymphoma, relapse, lenalidomide

ESHAP chemotherapy regimen associated to lenalidomide induces complete isotopic remission in Hodgkin's lymphoma relapsing after autologous stem cell transplantation

Dear Editor, Dear Reviewer, You can find here the suggested list of changes:

1. The time to relapse after BEAM was added as a new column at table one 2. Evaluation of responses was made by combined CT and 18F-FDG PET scan, using the International Harmonisation Project Criteria Dear Editor, Hodgkin's lymphoma is a rare human cancer characterised by a high rate of curability during the early stages of the disease. Despite the good results obtained with chemotherapy and radiotherapy, patients with advanced disease have a poor prognosis, especially those who relapse after autologous stem cell transplantation. These patients are rarely cured by a classical regimen of chemo-or radiotherapy. Several new strategies and drugs are under investigation based on the newest discoveries in disease biology. We treated three Hodgkin patients who had relapsed after high-dose chemotherapy and autologous stem cell transplantation with ESHAP (Etoposide, Cytarabin, Cisplatin and Methylpredisolone) chemotherapy and Lenalidomide, and their response was evaluated by 18F-FDG PET scan after three cycles. Because three complete isotopic responses were obtained, these patients could continue their therapeutic program via allogenic stem cell transplantation.

During a period of one year, we identified three Hodgkin's lymphoma patients who relapsed after autologous stem cell transplantation. The median age of the patients was 36 years. The characteristics of these three patients are presented in table 1

RxT -Radiotherapy

Table 1 -Characteristics of patients with Hodgkin's lymphoma relapsing after ABSCT and treated with ESHAP -Revlimid and the toxic effects of the regimen Each patient was heavily pre-treated before and after relapse with at least four lines of chemoand radiotherapy. The patients were treated with an ESHAP chemotherapy regimen once every three weeks and with Lenalidomide 15 mg daily. Lenalidomide was selected because of its anti-angiogenic effects as well as its abilities to induce apoptosis via p21 and cell cycle arrest and recruit effector T and NK cells. The patients gave their informed consent to be treated with Lenalidomide. On day 8 of each course of chemotherapy, all patients received primary G-CSF prophylaxis in a treatment regimen of 5 µg/kg/day for five days to prevent neutropenia. The adverse effects of this regimen were grade 4 neutropenia despite the presence of G-CSF and thrombocytopenia that required platelet transfusion. Eighteen days after the end of the third (second in one patient) cycle of chemotherapy, the patient response was evaluated by combined CT and 18F-FDG PET scan, using the International Harmonisation Project Criteria, and three complete isotopic responses were observed.

Because the good responses obtained, the patients could continue the therapeutic program and became candidates for allogenic stem cell transplantation in good conditions.

Hodgkin's lymphoma is a highly curable disease with a majority of patients presenting in early stages of disease and responding to the gold standard, anthracyclin-based ABVD chemotherapy [START_REF] Canellos | Chemotherapy of advanced Hodgkin's disease with MOPP, ABVD, or MOPP alternating with ABVD[END_REF][START_REF] Canellos | Long-term follow-up of Hodgkin's disease trial[END_REF]. Patients with an advanced stage of the disease have an high failure rate using standard therapy (3); these patients require more intensive strategies, most of which are based on high-dose chemotherapy, with or without autologous stem cell support (4,5,6,7). For those patients relapsing after the first line of chemotherapy, several regimens have been proposed, all of which were followed by ABSCT (autologous blood stem cell transpaltation). Those late-stage patients who relapse after ABSCT have a very poor prognosis. The majority of these patients underwent allogenic stem cell transplantation because of their young age [START_REF] Moreau | Successful allogeneic bone marrow transplantation for early relapse after autologous bone marrow transplantation in two cases of aggressive high-grade non-Hodgkin's lymphoma[END_REF][START_REF] Alvarez | Nonmyeloablative stem cell transplantation is an effective therapy for refractory or relapsed hodgkin lymphoma: results of a spanish prospective cooperative protocol[END_REF]. Unfortunately, there is no standard therapy for patients who relapse after ABSCT and will subsequently undergo AlloSCT. New discoveries leading to a better understanding of Hodgkin's disease has allowed new treatment strategies to be developed [START_REF] Younes | Novel treatment strategies for patients with relapsed classical Hodgkin lymphoma[END_REF]. One compound that is being tested is Lenalidomide, a powerful Thalidomide analogue. Previously, Reed-Sternberg cells were shown to be dependent on their microenvironment such that their culture is very difficult once removed from their "home" [START_REF] Kotla | Mechanism of action of lenalidomide in hematological malignancies[END_REF]. Lenalidomide has several effects on the tumour microenvironment and its growth: antiangiogenic effects, immunomodulation via alterations in cytokine production, activation of T-cells, augmentation of NK cell activity and direct anti-tumour activity by cell cycle arrest and the induction of apoptosis [START_REF] Fehniger | A phase 2 multicenter study of lenalidomide in relapsed or refractory classical Hodgkin lymphoma Blood[END_REF]. Recently, two trials used Lenalidomide as a single agent to treat patients with refractory or relapsed Hodgkin disease, with interesting results. Some patients responded to the treatment, and others experienced a stabilisation of their disease [START_REF] Boll | Lenalidomide in refractory or multiple relapsed Hodgkin lymphoma[END_REF]14). In our heavily pre-treated patients, ESHAP chemotherapy, in conjunction with Lenalidomide treatment, resulted in complete isotopic remission. In addition, the side effects were acceptable, which allowed the patients to remain in good condition while they continued their therapeutic program along with the allogenic stem cell transplantation.

  .

	Patie	Age Time to	Initial	Initial	Nr of	Stage at	line	Anemia	Neutropenia	Thrombocytopenia
	nt		relapse	stage	treatment	cycles	relapse	of
			after			ESHAP		TT
			BEAM			Rev
			(months)
	1	37	42	4	2ABVD,	2	2	4	grade 2	grade 3	grade 3
					2
					MOPPBVD
					BEAM
	2	40	3	4	2ABVD+	3	4	5	grade 3	grade 4	grade 4
					3VABEM+
					BEAM+
					RxT
	3	31	30	2	6ABVD+	3	4	7	grade 3	grade 4	grade 4
					2VABEM+
					1MINE+
					BEAM+
					RxT+
					4MINE+
					8MOPP

ABVD -Adriamycin, Bleomycin, Vindesin, Dacarbazin MOPP -Mechloretamin, Procarbazin, Vincristin, Prednison VABEM -Vindésin, Adriamycin, BCNU, Etoposid, Méthylprednisolon BEAM -BCNU, Etoposid, ARA-C, Melphalan MINE -Navelbine, Ifosfamide, Mitoguazone, Etoposide .


Ellen Ritter

Ralf A Husain

Katrin Hinderhofer

Tino Prell

Hans-Jörg Fricke

Sebastian Scholl

Andreas Hochhaus

Paul La Rosée
email: paul.larosee@med.uni-jena.de

Med P La Rosée

Ornithine transcarbamylase (OTC) deficiency based on a hemizygous p.R277W mutation causing life threatening hyperammonemic crisis during treatment for Hodgkin's lymphoma

Keywords: ornithine transcarbamylase deficiency, hyperammonemia, corticosteroids, Hodgkin's lymphoma

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Dear Editor,

We report on a rare case of ornithine transcarbamylase (OTC) deficiency as origin for life threatening hyperammonemia in a young adult male patient with Hodgkin's lymphoma.

Clinical case report

The reported patient was diagnosed with mediastinal Hodgkin's lymphoma (Ann Catalyzing the carbamylation of citrulline, mitochondrial OTC is crucially involved in ureagenesis and ammonia elimination. Currently, more than 300 mutations compromising enzyme activity are known [START_REF] Yamaguchi | Mutations and polymorphisms in the human ornithine transcarbamylase (otc) gene[END_REF]. OTC deficiency is an X-linked disorder [START_REF] Lindgren | Human ornithine transcarbamylase locus mapped to band xp21.1 near the duchenne muscular dystrophy locus[END_REF] that represents the most frequent inherited urea cycle defect. Hemizygous males with complete OTC deficiency display severe hyperammonemia within the first week(s) of life. Although males and heterozygous females with subtotal defects frequently present later, adult-onset OTC deficiency is rare [START_REF] Rohininath | Fatal presentation of ornithine transcarbamylase deficiency in a 62-year-old man and family studies[END_REF][START_REF] Harada | Late-onset ornithine transcarbamylase deficiency in male patients: Prognostic factors and characteristics of plasma amino acid profile[END_REF][START_REF] Atiq | Adult onset urea cycle disorder in a patient with presumed hepatic encephalopathy[END_REF] and found mostly in females with unfavorable lyonization.

Arbor
Several environmental factors have been implicated as potential triggers of late-onset OTC deficiency. These include valproate [START_REF] Thakur | Fatal cerebral edema from late-onset ornithine transcarbamylase deficiency in a juvenile male patient receiving valproic acid[END_REF], gastrointestinal bleeding [START_REF] Trivedi | Ornithine transcarbamylase deficiency unmasked because of gastrointestinal bleeding[END_REF], parenteral nutrition [START_REF] Felig | Hyperammonemic coma due to parenteral nutrition in a woman with heterozygous ornithine transcarbamylase deficiency[END_REF] and possibly corticosteroids [START_REF] Atiq | Adult onset urea cycle disorder in a patient with presumed hepatic encephalopathy[END_REF][START_REF] Summar | Unmasked adult-onset urea cycle disorders in the critical care setting[END_REF]. In the present case hyperammonemic encephalopathy and steroid administration showed correlation, and, in addition, a corticosteroid-dose dependence of the clinical manifestations was evident (Fig. 1).

Notably, chemotherapy without corticosteroids did provoke neither hyperammonemia nor encephalopathy. This suggests that both episodes of hyperammonemic encephalopathy resulted from a steroid-induced excessive protein catabolism on the background of a pre-existent reduction in the capacity of ammonia clearance. The latter was found to result from a specific mutation in the ornithine transcarbamylase (OTC) gene (p.R277W) previously shown to be associated with late disease onset [START_REF] Tuchman | The biochemical and molecular spectrum of ornithine transcarbamylase deficiency[END_REF]. Further genetic testing revealed the same mutation in the patient's mother and his thirteen-year-old brother both of whom were asymptomatic.

In conclusion, the reported case illustrates that late-onset OTC deficiency should be considered in hyperammonemic patients treated with steroids. Genotyping is essential for diagnosis, as our patient did not show elevated orotic acid commonly assumed to be central for the diagnosis of OTC deficiency.

Figure legend

  Ornithine transcarbamylase (OTC) deficiency based on a hemizygous p.R277W mutation causing life-threatening hyperammonemic crisis during treatment for Hodgkin's lymphoma Ellen Ritter, Ralf A. Husain, Katrin Hinderhofer, Tino Prell, Hans-Jörg Fricke, Sebastian Scholl, Andreas Hochhaus, Paul La Rosée To cite this version: Ellen Ritter, Ralf A. Husain, Katrin Hinderhofer, Tino Prell, Hans-Jörg Fricke, et al.. Ornithine transcarbamylase (OTC) deficiency based on a hemizygous p.R277W mutation causing life-threatening hyperammonemic crisis during treatment for Hodgkin's lymphoma. Annals of Hematology, 2010, 90 (7), pp.857-859. 10.1007/s00277-010-1106-y. hal-00615407

  stage IA) at the age of 23 years in November 2008. He underwent initial remission induction with chemotherapy (doxorubicin, bleomycin, vinblastine, dacarbazine) and involved field irradiation. In April 2009, early relapse (stage IIA) caused upper airway obstruction. Immediate treatment with intravenous prednisolone (continued with methylprednisolone p.o.) and irradiation was initiated. Four weeks later the patient presented with reduced vigilance finally progressing to coma. Blood testing revealed markedly elevated plasma ammonia levels (>400 µmol/l, reference range [RR] 16-48 µmol/l; Fig. 1); bilirubin and aminotransferases were marginally elevated. Electroencephalography displayed a moderately severe general alteration suggestive of metabolic encephalopathy. Cranial magnetic resonance imaging (MRI) showed slight cerebral edema and bilateral subcortical FLAIR hyperintensities in the precentral region with patchy distribution. Cerebrospinal fluid analysis, liver MRI and histology, drug and medication screening as well as microbiological investigations were negative. Corticosteroids were discontinued and the patient recovered completely without extensive anti-hyperammonemic therapy. In June 2009, chemotherapeutic treatment of relapse was initiated with dexamethasone, cytarabine and cisplatin (DHAP), and switched to the Dexa-BEAM regimen (dexamethasone, carmustine, etoposide, cytarabine, melphalan) due to refractory disease. Twelve days after the onset of Dexa-BEAM the patient again presented with nausea, vomiting, and progressive loss of vigilance. He was transferred to the intensive care unit for transient mechanic ventilation. Blood testing revealed severe hyperammonemia (Fig. 1), reduced citrulline (13 µmol/l, RR 19-55 µmol/l) and slightly elevated alanine levels (622 µmol/l, RR 241-597 µmol/l) pointing to a possible block of ureagenesis at the level of or proximal to OTC. Other plasma amino acids (including arginine and glutamine) and urinary orotic acid were in the reference range. Blood glucose, lactate, carnitine, acylcarnitines and urinary organic acids were normal. Accordingly, severe hepatic failure, organoacidurias and disorders of fatty acid oxidation appeared unlikely. Molecular analysis revealed a point mutation in the OTC gene leading to an exchange of arginine by tryptophan at position 277 (p.R277W mutation). Encephalopathy resolved within two days and further chemotherapy was continued without dexamethasone. In November 2009 the patient received autologous peripheral blood stem cell transplantation after a single dose of prednisolone (100 mg i.v.). Treatment was well tolerated and hyperammonemic encephalopathy did not re-occur. In January 2010, however, the patient died from infectious complications.

Fig. 1

 1 Fig. 1 Correlation of hyperammonemia and encephalopathy with corticosteroid


MD Daan Dierickx
email: daan.dierickx@uzleuven.be

Gregor Verhoef

André Delannoy

Rituximab in patients with refractory autoimmune hemolytic anemia

published or not. The documents may come

Dear Editor,

We have read with great interest the article by Penalver et al in which they report the results of their Spanish multicenter retrospective analysis of 36 cases of refractory autoimmune hemolytic anemia treated with rituximab. In their analysis the authors show that treatment with anti CD20 monoclonal antibodies is associated with an overall and complete response rate of 77% and 61% respectively [START_REF] Penalver | Rituximab is an effective and safe therapeutic alternative in adults with refractory and severe autoimmune hemolytic anemia[END_REF]. As stated by Penalver et al most available evidence on the use of rituximab in AIHA is based on retrospective case reports and smaller case series, with only few uncontrolled prospective trials, which is mainly due to the rarity of the disorder.

We were surprised by the fact that the authors did not refer to and make a comparison with our Belgian retrospective analysis, which was published in 2009 in the Journal of Internal Medicine. In this study, which had a very similar design as the study of Penalver et al, we reported on 53 patients with refractory AIHA treated with rituximab. Compared with the Spanish results overall response rates (79%) were similar, although the rate of complete responses (CR) was lower (47%) [START_REF] Dierickx | Rituximab in auto-immune haemolytic anaemia and immune thrombocytopenic purpura: a Belgian retrospective multicentric study[END_REF]. The reason for this difference is not clear as definition of CR and proportion of patients with cold type AIHA, a subtype in which the achievement of CR with rituximab monotherapy is rather rare, were similar [START_REF] Berentsen | Rituximab for primary chronic cold agglutinin disease: a prospective study of 37 courses of therapy in 27 patients[END_REF]. In our study we were not able to identify pretreatment characteristics predictive for response or for response duration, including prior splenectomy. In contrast, patients with prior splenectomy showed better response rates than non-splenectomized patients in the Spanish study. Interestingly, in another retrospective analysis examining the use of rituximab in immune mediated thrombocytopenia (ITP), Penalver et al reported a higher early response rate in non-splenectomised patients, but no difference in maintained response. The authors suggested that an intact spleen might be necessary to achieve an early response [START_REF] Penalver | Rituximab in the management of chronic immune thrombocytopenic purpura: an effective and safe therapeutic alternative in refractory patients[END_REF]. The importance of prior splenectomy with regard to response rates in both immune mediated disorders remains a matter of debate, with conflicting results in various series. Penalver et al also discussed the issue of retreatment with rituximab in patients previously responding to the antibody. In our series nine patients were re-challenged with rituximab, leading to overall response characteristics similar to the responses achieved after the first course, including in some cases even longer response duration after rituximab re-treatment. In addition, one patient achieved a partial response although first rituximab course failed [START_REF] Dierickx | Rituximab in auto-immune haemolytic anaemia and immune thrombocytopenic purpura: a Belgian retrospective multicentric study[END_REF].

In conclusion, AIHA is a rare disorder in which relapses are common necessitating new therapeutic options. Rituximab may be an important alternative for the conventional therapies of these disorders, either in monotherapy or in association with other forms of immunotherapy [START_REF] Berentsen | High response rate and durable remissions following fludarabine and rituximab combination therapy for chronic cold agglutinin disease[END_REF].


Guillaume Lefevre

Jean-Christophe Ianotto
email: jcianotto@hotmail.com

Adrian Tempescul

Philippe Lemoine

Gaelle Guillerm

Christian Berthou

Infection by H1N1 flu virus revealing T-cell acute lymphoid leukaemia: About two cases

Keywords: H1N1 influenza virus, oseltamivir, T-cell acute lymphoid leukaemia, lymphopenia

Dear Editor,

We present here two exceptional cases of young patients who developed pulmonary infections due to the H1N1 flu, which revealed the presence of T-acute lymphoid leukaemia. In late 2009, a 16-year-old woman, and two weeks later, a 12-year-old boy were admitted to our hospital for pulmonary syndrome associated with high fever. The clinical presentation was similar for both patients: asthaenia, high fever, cough, rhinorrhea and dyspnoea, but no thoracic pain. The two patients needed oxygen therapy. Chest x-rays of both patients showed bilateral interstitial syndrome and right basal condensation. CT scans confirmed the bilateral alveolar condensation syndrome with an air bronchogram, ground glass opacities and numerous fuzzy nodules (Figure 1). The two CT scans revealed multiple mediastinal adenopathies and a large thymus. The H1N1 flu virus was suspected because of the epidemic in the winter of 2009. The diagnosis was confirmed by real-time PCR using the H1SWL test (M-CNR Pasteur gene) with the Lightcycler 2.0 (Roche) and broncho-alveolar lavage fluid from the girl girland nasal liquid from the boy. Due to the high fever and neutropaenia, the patients were immediately treated with antibiotics and cefotaxime (ciprofloxacine and cefotaxime for the girl; cefotaxime, josamycin and amikacin for the boy), but with little success. After the diagnosis of H1N1 flu infection, we used oseltamivir at the recommended dose of 75 mg per day (from day 3 for the first patient and day 1 for the boy). The symptoms disappeared a few days after beginning the oral treatment, and oxygen was no longer needed.

Oseltamivir treatment was stopped after 5 days.

During this time, we observed cytopaenias in the haemograms of both patients. For the girl and boy, the haemoglobin levels were 77 and 44 g/L, platelets were 224 and 61 x10 9 /L, white blood cells were 17.1 and 1.9 x10 9 /L, and neutrophils polymorphonuclear were 0.34 and 0.42 x10 9 /L, respectively. Blood exams showed the presence of medium-size blasts quantified at 14.5 and 0.8 x10 9 /L for the girl and boy, respectively. We observed 3% of circulating CD4+ T-cell lymphocytes for the girl and none for the boy. The LDH levels were 585 and 728 UI/L, respectively. C-reactive protein was 34 and 202 mg/L. The diagnosis of T-ALL was made based on examination of the bone marrow: the infiltration of blasts was higher than 90%, with sizes ranging from medium to large; the nucleo-cytoplasmic ratio was elevated; the cytoplasm was hyperbasophil with no granulations. Examination of the blasts using flow cytometry confirmed that they were of T-cell origin. The karyotypes were normal. The final diagnosis of type II T-ALL was made for both patients. When the pulmonary symptoms disappeared, we introduced chemotherapy using the same induction therapy for both patients: corticosteroids alone for the first week and thereafter corticosteroids together with vincristine, daunorubicine, cyclophosphamide and asparaginase. Intra-thecal injections of chemotherapy were given 3 times during induction, and no meningeal localisation of virus was observed. The presence of the H1N1 influenza virus did not influence the progress of the chemotherapy protocol. The girl was resistant to corticosteroids at day 8 and to chemotherapy at day 15. The boy was sensitive to both corticosteroids and chemotherapy. The two patients were in complete haematological remission at the end of the induction phase. They followed their therapy, and no additional lung infections occurred. They are still in complete remission and in treatment today.

The cases described here are exceptional because in all previously reported cases of H1N1 influenza in leukemic patients, the virus infection appeared during the aplastic phase secondary to chemotherapy. There is only one reported case of the H1N1 flu in a patient with hemopathy: a 67-year-old man who developed an H1N1 infection during a chemotherapy (cyclophosphamide, vincristine and prednisolone) treatment for CLL and died after 4 weeks [START_REF] Van Der Vries | Fatal oseltamivir-resistant influenza virus infection[END_REF].

In the cases reported here, the two patients had deep lymphopaenia at the time of diagnosis, which was probably present for many weeks. The first sign of T-ALL was the presence of an H1N1 flu virus pulmonary infection, which was observed during the winter pandemic of this virus. The association between lymphopaenia and the H1N1 flu virus has been previously shown, with a higher mortality in the patients with low count. No secondary infections were observed, and neither patient experienced ARDS, perhaps due to the neutropaenia, despite the fact that previously reported deaths were mostly due to ARDS [START_REF] Perez-Padilla | INER Working Group on Influenza. Pneumonia and respiratory failure from swine-origin influenza A (H1N1) in Mexico[END_REF][START_REF] Laufe | Adult respiratory distress syndrome in neutropenic patients[END_REF]. The only recommended treatment for the H1N1 virus is oseltamivir, and this treatment is highly effective [START_REF] Hajjar | The first influenza pandemic of the 21st century[END_REF][START_REF] Maritz | Pandemic influenza A (H1N1) 2009: the experience of the first six months[END_REF][START_REF] Sullivan | H1N1 influenza[END_REF][START_REF] Gordon | Update on 2009 pandemic influenza A (H1N1) virus[END_REF]. We confirmed the efficacy in immuno-compromised patients. In these two cases, the occurrence of the H1N1 flu virus did not result in modification of the treatment nor the results of the chemotherapy as the two patients were alive 7 months after the diagnosis and in complete haematological remission. These cases suggest that H1N1 virus infections could be masking other conditions in immuno-compromised patients, and they confirm that oseltamivir is effective in these patients.

No conflict of interest.

Figure 1

 1 Figure 1 CT scan confirmed the bilateral alveolar condensation syndrome with an air bronchogram, ground glass opacities and numerous fuzzy nodules.


MD Dries Deeren
email: ddeeren@hhr.be

A kidney transplant patient with pure red cell aplasia: first things first!

Keywords: Allopurinol Kidney failure, chronic Kidney transplantation Red-cell aplasia, pure

Letter to the Editor Acquired pure red cell aplasia (PRCA) is a rare cause of anemia characterized by the virtual absence of erythroid precursors in the bone marrow. The other cell lineages are normal.

Adult acquired PRCA is most often idiopathic, although an underlying cause can be found in some patients. Large granular lymphocyte leukemia, myelodysplasia, thymoma, chronic lymphocytic leukemia, allogeneic stem cell transplantation, anti-erythropoietin antibodies secondary to treatment with recombinant human erythropoietin, drugs, viral infections and autoimmune diseases have all been associated with PRCA [START_REF] Fisch | Pure red cell aplasia[END_REF].

One of the most important aspects of initial management is the cessation of possible offending drugs, although only a minority of patients will respond to this approach alone.

However, when PRCA is diagnosed in a kidney transplant patient, several of the possible causes may seem more likely, and this vital step in the treatment is forgotten.

In April 2010 a 53-year-old kidney transplant patient was referred because of anaemia, not responsive to darbepoietin. Darbepoietin was started because of this anaemia. The hemoglobin had been decreasing for more than one year, and had reached 6.5 g/dL. The rest of the blood count was unremarkable. The creatinine level was stable at 2.2 mg/dL. There were no signs of haemolysis nor bleeding, and upper and lower gastrointestinal endoscopy revealed no abnormalities. There was no reticulocytosis, vitamin or iron deficiency. Bone marrow examination showed as the only abnormality the absence of red cell precursors, compatible with PRCA.

An evaluation for the classical causes of PRCA was negative, except for one iatrogenic cause. The patient had been taking allopurinol at the relatively high dose of 300 mg/dayconsidering the kidney function -for several years before the hemoglobin level started to decrease. Allopurinol was stopped immediately and packed cells were transfused. Very soon, many reticulocytes were released in the peripheral blood and the hemoglobin level rose (Fig 1). Allopurinol was never reintroduced. Five months later, the hemoglobin level stays normal, and there have been no other health problems.

Although there are reports on allopurinol-induced PRCA [START_REF] Chao | Hypersensitivity syndrome and pure red cell aplasia following allopurinol therapy in a patient with chronic kidney disease[END_REF][START_REF] Lin | Acute pure red cell aplasia associated with allopurinol therapy[END_REF][START_REF] Shankar | Allopurinol-induced pure red cell aplasia[END_REF], this cause of PRCA is relatively unknown. This case illustrates that simple causes have to be ruled out first, even in complex patients after kidney transplantation. There is no standard treatment for PRCA except for red cell transfusions, although several drugs have been reported to be effective in selected cases [START_REF] Fisch | Pure red cell aplasia[END_REF]. Cessation of the offending drug is not only of critical importance, but also the easiest, least expensive and one of the few evidence based treatment options. This case also demonstrated that the indication and dosage adjustment should be carefully evaluated before starting allopurinol therapy in patients with chronic kidney disease. Evolution of the hemoglobin level from December 1 st until June 1 st 2010. The last dose of allopurinol was taken on April 7 th 2010.

Figures

Figure

  Figure 1

1

 1 Figure 1


Gabriele Buda

Pegylated Liposomal Doxorubicin in combination with Dexamethasone and Bortezomib (VMD) or Lenalidomide (RMD) in Multiple Myeloma pretreated patients

Keywords: myeloma, liposomal doxorubicin, bortezomib, lenalidomide

Waterman et al recently showed that a modified regimen of pegylated liposomal doxorubicin (PLD), bortezomib, and dexamethasone is effective and well tolerated in the treatment of relapsed or refractory multiple myeloma [START_REF] Waterman | A modified regimen of pegylated liposomal doxorubicin, bortezomib, and dexamethasone is effective and well tolerated in the treatment of relapsed or refractory multiple myeloma[END_REF]. We really appreciated these data that were similar to results that we already described in similar patients [START_REF] Buda | Bortezomib and liposomal doxorubicin are highly effective in obtaining the best possible response before autologous transplant for multiple myeloma[END_REF]. We treated a total of 25 patients (11 females and 14 males) with a median age of 61 years at diagnosis (range: 40-69) who were resistant/relapsed after at least one prior therapy. The scheme therapy was based on VMD: bortezomib: 1. In our study, lenalidomide or bortezomib in combination with PLD and dexamethason has shown encouraging activity in heavily pretreated patients with relapsed or refractory multiple myeloma. These schemes can be additional standard of care in the treatment of patients with relapsed or refractory multiple myeloma who have received at least one prior therapy (3). The addition of PLD can play a key role in overcoming anthracycline resistance and improving the quality of response without limiting toxicity. The pharmacology of PLD gives rise to a compound with major advantages that could potentially improve response and decrease toxicity. Because increased angiogenic activity occurs in the bone marrow of patients with multiple myeloma, this pegylated formulation can enhance the delivery of doxorubicin to the tumour site. In addition, because myeloma cells divide slowly, the increased exposure of these cells to doxorubicin has the potential of overcoming resistance and increasing tumour cell killing capacity, theoretically resulting in improved response rates (4).

Liposomal Doxorubicin in combination with Dexamethasone and Bortezomib (VMD) or Lenalidomide (RMD) in Multiple Myeloma pretreated patients

	Manuscript
	Click here to download Manuscript: buda.annals.11010R1.doc	Click here to view linked References
	1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9	Pegylated Gabriele Buda * , Enrico Orciuolo * , Sara Galimberti , Matteo Pelosini , Mario Petrini
	10 11 10 11	* These authors contributed equally to this work
	12 12
	13 14 13 14	Dept. of Oncology, Transplants and Advanced Technologies, Section of Hematology, Pisa University, Italy
	15 15
	16 16	Corresponding Author: Dr. Gabriele Buda, Section of Hematology, Pisa University, via Roma 67, 56100
	17 17	Pisa, Italy
	18 18
	19 20 21 22 19 20 21 22	Email ga.buda@libero.it Tel 0039 050 992755 FAX 0039 050 992903
	23 23
	24 24
	25 25
	26 26
	27 27
	28 28
	29 29
	30 30
	31 31
	32 32
	33 33
	34 34
	35 35
	36 36
	37 37
	38 38
	39 39
	40 40
	41 41
	42 42
	43 43
	44 44
	45 45
	46 46
	47 47
	48 48
	49 49
	50 50
	51 51
	52 52
	53 53
	54 54
	55 55
	56 56
	57 57
	58 58
	59 59
	60 60
	61 61
	62 62
	63 63
	64 64
	65 65

Table I .

 I Characteristics of MM Patients undergoing VMD or RMD therapy

	1
	2 2
	3 4 5 3 4 5	Characteristics Age at diagnosis (median and range)	Cases 65 (44-76)
	6 6
	7 7	Number of patients
	8 9 10 8 9 10	VMD RMD	25 (14M, 11F) 14 (10M, 4F)
	11 11
	12 12	Stage at diagnosis
	13 14 13 14	Durie-Salmon (II/III)	10/29
	15 16 17 15 16 17	Prognostic Markers 2-microglobulin (/L.)	2.2 (1.1 -35) a
	18 18	Creatinin (mg/dl.)	0.9 (0.5 -4.4) a
	19 20 21 19 20 21	Albumin (g/dl)) Hemoglobin (mg/dl)	4.0 (2.1 -4.9) a 11.3 (5.7 -16.4) a
	22 22
	23 23	. a Median (Range)
	24 24
	25 25
	26 26
	27 27
	28 28
	29 29
	30 30
	31 31
	32 32
	33 33
	34 34
	35 35
	36 36
	37 37
	38 38
	39 39
	40 40
	41 41
	42 42
	43 43
	44 44
	45 45
	46 46
	47 47
	48 48
	49 49
	50 50
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Dr Kevin Kemp
email: kevin.kemp@bristol.ac.uk

Dr Ruth Morse

Dr Kelly Sanders

Professor Jill Hows

Dr Craig Donaldson

M S Group

Alkylating chemotherapeutic agents cyclophosphamide and melphalan cause functional injury to human bone marrow-derived mesenchymal stem cells

Keywords: Mesenchymal Stem Cells, Transplantation, Chemotherapy, Hematopoietic Stem Cells, Bone marrow

HAL is

Introduction

The biological functions of bone-marrow derived mesenchymal stem cells (MSCs) in vivo include both hematopoietic support and tissue maintenance. These functions are achieved through MSCs having a multipotent capability to generate progeny that can differentiate down multiple cell lineages to form bone, cartilage, fat cells, and bone-marrow stroma [START_REF] Pittenger | Multilineage potential of adult human mesenchymal stem cells[END_REF], in addition to having the apparent ability to support the expansion of primitive hematopoietic cells through the expression of a variety of cytokines and the reconstruction of the hematopoietic microenvironment [START_REF] Rafii | Regulation of hematopoiesis by microvascular endothelium[END_REF][START_REF] Majumdar | Human marrowderived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages[END_REF][START_REF] Majumdar | Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells[END_REF].

Hematopoietic recovery after high dose chemotherapy (HDC) in the treatment of hematological diseases may be slow and/or incomplete. This is generally attributed to progressive hematopoietic stem cell failure, although defective hematopoiesis may be in part due to poor stromal function. HDC and irradiation used with or without autologous hematopoietic stem cell (HSC) rescue in the treatment of hematological malignancies and other cancers may cause long lasting damage to bone marrow stromal cells, thus impairing hematopoiesis and may have a possible involvement in slow or poor engraftment post HSC transplantation.

Patients who have undergone HDC commonly display disruption of the marrow architecture with hemorrhaging, loss of fat, and loss of stromal compartments [START_REF] Ishida | Requirement of donor-derived stromal cells in the bone marrow for successful allogeneic bone marrow transplantation. Complete prevention of recurrence of autoimmune diseases in MRL/MP-Ipr/Ipr mice by transplantation of bone marrow plus bones (stromal cells) from the same donor[END_REF]. Studies have also demonstrated that a recipient's stromal cells are damaged after allogeneic stem cell transplantation [START_REF] Domenech | Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation[END_REF][START_REF] Galotto | Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients[END_REF][START_REF] O'flaherty | Bone marrow stromal function from patients after bone marrow transplantation[END_REF], or even from chemotherapeutic drugs alone [START_REF] Domenech | Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation[END_REF][START_REF] Galotto | Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients[END_REF][START_REF] O'flaherty | Bone marrow stromal function from patients after bone marrow transplantation[END_REF][START_REF] Tabilio | Effect of chemotherapy for acute myelogenous leukemia on hematopoietic and fibroblast marrow progenitors[END_REF][START_REF] Corazza | Bone marrow stroma damage induced by chemotherapy for acute lymphoblastic leukemia in children[END_REF][START_REF] Banfi | High-dose chemotherapy shows a dose-dependent toxicity to bone marrow osteoprogenitors: a mechanism for post-bone marrow transplantation osteopenia[END_REF][START_REF] Cohen | Effect of chemotherapy and irradiation on interactions between stromal and hemopoietic cells in vitro[END_REF][START_REF] Domaratskaia | Alkylating damage by dipin of hematopoietic and stromal cells of the bone marrow[END_REF][START_REF] Domenech | Haemopoiesis of transplanted patients with autologous marrows assessed by long-term marrow culture[END_REF][START_REF] Fried | Effects of radiation on hematopoietic stroma[END_REF].

We have also recently demonstrated a significant reduction in MSC expansion and MSC CD44 expression by MSCs derived from patients receiving HDC regimens for hematological malignancies, thus implicating potential disadvantages in the use of autologous MSCs in chemotherapeutically pre-treated patients for future therapeutic strategies [START_REF] Kemp | Chemotherapyinduced mesenchymal stem cell damage in patients with hematological malignancy[END_REF]. These effects are relevant not only in patients treated with HDC without allogeneic stem cells but also in recipients of allogeneic stem cell transplants in whom bone marrow stroma remains recipient in origin after the transplant [START_REF] Simmons | Host origin of marrow stromal cells following allogeneic bone marrow transplantation[END_REF][START_REF] Cilloni | Limited engraftment capacity of bone marrow-derived mesenchymal cells following T-cell-depleted hematopoietic stem cell transplantation[END_REF][START_REF] Koc | Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases[END_REF]. If damage caused by chemotherapeutic agents to marrow MSCs is substantial, it would be logical to use cultured allogeneic MSCs therapeutically to assist or repair the marrow microenvironment after HDC. Evidence from animal experiments [START_REF] Fibbe | Ex vivo expansion and engraftment potential of cord blood-derived CD34+ cells in NOD/SCID mice[END_REF][START_REF] Almeida-Porada | Cotransplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero[END_REF][START_REF] Angelopoulou | Cotransplantation of human mesenchymal stem cells enhances human myelopoiesis and megakaryocytopoiesis in NOD/SCID mice[END_REF][START_REF] Anker | Nonexpanded primary lung and bone marrow-derived mesenchymal cells promote the engraftment of umbilical cord blood-derived CD34(+) cells in NOD/SCID mice[END_REF] and clinical trials [START_REF] Koc | Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy[END_REF] suggests that co-transplantation of cultured allogeneic MSCs may have a role in facilitating hematopoietic stem cell engraftment after stem cell transplants, although the biological mechanisms involved are unclear. and differences in the drug combinations used, both of which could have implications on MSC function. With drugs not being commonly used as single agents during HDC regimens there is also a lack of data comparing the short or long term effects that these drugs have on patients post treatment. This in-vitro study was therefore designed to help address these problems using the alkylating agents cyclophosphamide and melphalan at clinically relevant concentrations to evaluate the effects of chemotherapeutic exposure in-vitro on the functional properties of femoral-shaft marrow MSC cultures.

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Methods

Femoral shaft bone marrow collection

Bone marrow samples were obtained by an Orthopaedic surgeon at the Avon Orthopaedic Centre (AOC), Southmead Hospital, Bristol, with informed written consent and hospital ethics committee approval. Bone marrow was removed from the femoral shaft during surgery for total hip replacement to make space for the prosthetic joint and placed into a sterile 50ml tubes containing 1000 I.U heparin. Patients with a history of malignancy, immune disorders or rheumatoid arthritis were excluded from the study. Femoral shaft bone marrow donors were healthy apart from osteoarthritis, and were not receiving drugs known to be associated with myelosuppression or bone marrow failure.

Umbilical cord blood sample collection

Umbilical cord bloods were obtained by midwives in the Central Delivery Suite, Southmead Hospital, Bristol, with maternal written informed consent and local hospital ethics committee approval. Cord blood samples were collected by gravity into sterile 50ml tubes containing 1000 I.U heparin after the umbilical cord had been clamped and cut. All samples were from normal full-term deliveries, and collection was entirely at the discretion of the midwife in charge.

Establishment of mesenchymal culture

Femoral shaft marrow samples were broken up with a scalpel and washed with Dulbecco's Modified Eagles Medium (DMEM) until remaining material (bone) looked white at the bottom of the 50ml tube. All washings were pipetted into a new 50ml tube and kept for centrifugation. The suspension was centrifuged and resuspended in DMEM. Marrow aspirates were overlaid onto an equal volume of Lymphoprep™ (Axis-Shield, Dundee, UK) (density 1.077+/-0.001g/ml) and centrifuged at 600g for 35mins at room temperature to separate the mononuclear cells (MNC) from neutrophils and red cells. The mononuclear cell (MNC) layer was harvested and washed twice in DMEM Sigma-Aldrich, UK).

MSC culture

Isolated MNCs were centrifuged and resuspended in MSC medium (DMEM with 10% FCS selected for the growth of MSCs (StemCell Technologies, London, UK), and 1% Penicillin and Streptomycin (Sigma-Aldrich, Gillingham, UK)). Vented flasks (25cm 2 ) containing 10ml of MSC medium were seeded with 1x10 7 nucleated cells (seeding density = 400,000 cells/cm 2 ) for passage 0. Flasks were incubated at 37ºC in a humidified atmosphere containing 5% CO 2 and non-adherent hematopoietic cells removed by media exchange after 3-5 days. Cells were then cultured for two weeks at passage 0 and fed by half medium exchange.

To calculate MSC expansion, the adherent cells were resuspended using 0.25% trypsin (Sigma-Aldrich, Gillingham, UK) and re-seeded at 7.5x10 4 cells per flask (seeding density = 3000 cells/cm 2 ) into passage 1.

Thereafter, the cells were re-plated at 7.5x10 4 cells per flask (seeding density = 3000 cells/cm 2 ) every 14 days for up to 5 passages. During this time cells were fed every week with MSC medium by half medium exchange.

Immunophenotyping MSC cultures

To ensure a homogenous population of MSCs had been cultured immunophenotyping of surface markers, using flow cytometric techniques, was carried out according to previous reports [START_REF] Pittenger | Multilineage potential of adult human mesenchymal stem cells[END_REF][START_REF] Wexler | Adult bone marrow is a rich source of human mesenchymal 'stem' cells but umbilical cord and mobilized adult blood are not[END_REF][START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF]. MSCs were examined using the fluorescently tagged monoclonal antibodies anti-CD105, -CD45 -CD166, -CD44, -CD29 (BD Biosciences, Oxford, UK). For immunophenotypic analysis, MSCs were detached from culture flasks at second passage using 0.25% trypsin for 5 min, washed with PBS to remove trypsin, and resuspended in MSC medium at 10 6 cells/ml. The cell suspension was incubated in the dark at 4°C for 30 minutes with the specific monoclonal antibody. Cells were then washed with DMEM, centrifuged at 400g for 5mins, and resuspended in MSC medium for analysis. At least 10,000 events were analyzed on a BD FACS vantage SE and analyzed with CellQuest™ software (BD Biosciences, Oxford, UK). Gates were set on the analysis to remove cellular debris.

Differentiation

Mesenchymal stem cells were induced into adipogenic, osteoblastic and chondrogenic differentiation by culturing identical numbers of MSCs, at second passage, in NH Adipodiff medium, NH Osteodiff medium and NH Chondrodiff medium (Miltenyi Biotec, Surrey, UK) respectively according to the manufacturer's instructions. Differentiation of MSCs was only performed prior to any in vitro treatment with drugs, as differentiation was used solely for the purpose of MSC characterisation. Adipogenic differentiation was visualized by the accumulation of lipid-containing vacuoles which stain red with oil red O. Osteogenic differentiation was visualized morphologically and also by the presence of calcium stained with Alizarin red.

Finally chondrogenic differentiation was characterised by the production of the extracellular matrix proteoglycan aggrecan, visualized using immunofluorescent detection by labeling of aggrecan using a mouse anti-human aggrecan (4F4) antibody (Santa Cruz Biotechnology, Heidelberg, Germany).

Chemotherapeutic agents

The following chemotherapeutic agents were tested: Melphalan (50 μmol/L) or Cyclophosphamide (500 μmol/L) with the addition of S9 extract (0.4mg/ml) (Sigma-Aldrich, UK). Cells prior to each experiment were incubated with each treatment for 48 hours in DMEM, 10% FCS. The concentrations used for each chemotherapeutic agent used were determined from published data of plasma concentrations from patients undergoing intensive high-dose chemotherapy treatment or pre-stem cell transplant conditioning [START_REF] Chen | Nonlinear pharmacokinetics of cyclophosphamide in patients with metastatic breast cancer receiving high-dose chemotherapy followed by autologous bone marrow transplantation[END_REF][START_REF] Li | Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents[END_REF][START_REF] Lazarus | Intensive melphalan chemotherapy and cryopreserved autologous bone marrow transplantation for the treatment of refractory cancer[END_REF][START_REF] Pinguet | Pharmacokinetics of high-dose intravenous melphalan in patients undergoing peripheral blood hematopoietic progenitor-cell transplantation[END_REF][START_REF] Alberts | Comparative pharmacokinetics of chlorambucil and melphalan in man[END_REF].

S9 extract contains the cytochrome P450 proteins that are an essential group of enzymes involved in the metabolism of drugs and chemotherapeutic agents [START_REF] Rooney | The role of cytochrome P450 in cytotoxic bioactivation: future therapeutic directions[END_REF]. Most cell cultures contain little, if any, cytochrome P450 mixed function oxidase metabolic capability [START_REF] Li | Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents[END_REF], therefore S9 extract was used as a supplement in culture as cyclophosphamide requires systematic bioactivation by cytochrome P450 into its active cytotoxic compound 4-hydroxycyclophosphamide which forms both phosphoramide mustard and acrolein [START_REF] Nieto | Pharmacokinetics of high-dose chemotherapy[END_REF]. The effects of S9 extract alone on experimental conditions was investigated in all cases and not shown to be significantly different from control conditions (data not shown).

Cytotoxicity assay

Cell viability after chemotherapeutic insult was detected using the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (Promega, UK), according to the manufacturer's instructions. Prior to treatment cells were plated at 750 MSCs/well in 100μl of MSC media and were dispensed into 96-well plates in triplicate and incubated at 37˚C in a humidified atmosphere containing 5% CO 2 until cultures had reached confluence.

Immunoblotting for MSC CD44

At 48 hours post treatment cells were lysed using Beadlyte cell signalling universal lysis buffer (Upstate™, UK). The 2-D Quant Kit™ (GE Healthcare) was then used to quantify the concentration of total protein within each cell lysate sample according to manufacturer's instructions to ensure equal loading of cell lysates. Lysates were heated to 95˚C for five minutes with Laemmli 2x sample buffer (Invitrogen, UK) and run on NuPAGE Novex 4-12% Bis-Tris Zoom gels (Invitrogen). After transfer to PVDF membrane (Bio-Rad, UK) and blocking in 5% w/v powdered milk, membranes were incubated overnight in primary antibody at 4˚C (in Tris-buffered saline/5% bovine serum albumin). The antibody used was CD44 antibody (BRIC235 obtained from the IBGRL, Bristol) diluted 1:1000 (v/v) in PBS-Tween, 5% BSA. Immunoreactivity was detected using secondary antirabbit horseradish peroxidase conjugated antibodies (Abcam, UK) (in Tris-buffered saline/5% bovine serum albumin) and specific protein expression patterns were visualized by chemiluminescence using an Amersham ECL Plus™ Western Blotting Detection System (Amersham, UK).

Purification and flow cytometric analysis of CD34 + cord blood cells

CD34 + cell isolation was undertaken after MNC separation as previously described in the establishment of MSC culture. CD34 + cells were isolated from the MNC fraction obtained from cord blood harvests using the immunomagnetic MiniMACS (Magnetic-Activated Cell Sorter) CD34 isolation system according to the manufacturer's instructions (Miltenyi Biotec, UK). The CD34 + cells obtained from the MACS positive fraction were then assessed by cell counting and flow cytometry as described in the 'assessment of peripheral blood contamination of marrow MNC harvests' section above, with the exception that cells were assessed for CD34 + content by labelling with anti-CD34 clone HPCA-2 (BD Biosciences, Oxford, UK). Only CD34 + events with low side scatter were counted as CD34 + cells. CD34 + cells were cryo-preserved in liquid nitrogen until use in long-term culture assay.

Long-term culture of CD34 + cells on MSC derived stromal layer

MSC cultures, at second passage, were plated into 25cm 2 vented flasks at 7.5x10 4 cells per flask (seeding density = 3000 cells/cm 2 ) in 5ml of long-term culture medium (LTCM) (IMDM (Sigma-Aldrich, Gillingham, UK) containing 10% FCS (StemCell Technologies, London, UK), 10% horse serum (StemCell Technologies, London, UK), hydrocortisone (5x10 -7 M) and 1% Penicillin/Streptomycin. Cells were fed every week with LTCM by half medium exchange. After three weeks long-term culture cells were treated with chemotherapeutic agents for 48hours. Cultures were then washed repeatedly to remove all traces of the chemotherapeutic agents and all experiments were seeded with identical cryo-preserved CD34 + cell populations derived from the same cord blood source (2.5 x 10 4 cells/flask (seeding density = 1000 cells/cm 2 )) in LTCM. Flasks were incubated at 37˚C and fed weekly with LTCM by half media exchanges. All media removed each week when feeding was assessed for the numbers of supernatant cells present and CFU-GM content using the CFU-GM assay described below.

CD34 + cells are unable to produce stroma under the culture conditions used so it can be assumed that stromal elements grown in culture were MSC-derived [START_REF] Hows | Growth of human umbilical-cord blood in longterm haemopoietic cultures[END_REF]. As flow cytometric analysis indicated that there was no significant contamination of MSC cultures with cells of hematopoietic origin, it was decided not to irradiate MSC cultures prior to CD34 + cell seeding as it was hypothesized that MSCs may have an altered sensitivity to radiation and DNA damage post chemotherapy exposure.

CFU-GM assay

Supernatant cells removed from long-term cultures each week were plated at a concentration of 10 4 cells/well (seeding density = 26316 cells/cm 2 ) into 0.25ml of MethoCult® GF H4434 (StemCell Technologies, London, UK) in triplicate into 12-well tissue culture plates. The number of hematopoietic colonies, which are derived from the CB CD34 + cell population, present within each well after two weeks culture at 37˚C in a humidified atmosphere containing 5% CO 2 was then assessed.

Human Umbilical Vein Endothelial Cell (HUVEC) culture

The HUVEC cell line was obtained from the European Collection of Cell Cultures (ECACC) and grown in 75cm 2 vented fibronectin coated culture flasks. Cells were seeded in fibronectin coated flasks at a concentration of 1 x 10 4 cells per cm 2 in Endothelial Cell Growth Medium (ECACC) and fed by half medium exchange every other day and trypsinised every week.

CD34 + cell transmigration assay

For transmigration experiments 1x10 4 HUVEC cells were seeded on human fibronectin coated 5μm Millicellpolyethylene terephthalate membrane hanging transwell inserts for 24-well plates (Milipore®). Inserts were prior incubated with 0.01mg of human fibronectin (Millipore) in 0.1ml of DMEM for approximately 1 hour.

After three days, the monolayers had reached confluence and were suitable for use within the assay. MSC cultures in 24-well plates were treated with cyclophosphamide or melphalan and incubated for 48 hours.

Cultures were washed repeatedly to remove all traces of the chemotherapeutic agents and suspended in 600μl of MSC media. The Millicell transwell membrane inserts containing a HUVEC monolayer attached to the upper membrane surface were added to the 24-well plates containing the MSC cultures. A total of 6 x10 4 CD34 + cells in 400μl of MSC media were added into the upper chamber of each transwell insert placed in the 24-well plate.

The transwell set-up thus consisted of an upper and lower chamber separated by an endothelial cell layer. After 24 hours, non-adherent CD34 + cells that had transmigrated the endothelial cell layer into the lower chamber were recovered and enumerated using a progenitor cell (CFU-GM) assay, as described above.

Cell cryopreservation and thawing

Cell counts were obtained and cell density adjusted to <1 × 10 7 cells/ml with DMEM supplemented with 20% FCS, to which an equal volume of DMEM/20% Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, UK) was added.

The vials were cooled until they had reached -80°C using a 5100 Cryo 1°C freezing container (Nalgene, DK).

Tubes were then transferred to a liquid nitrogen container for permanent storage until use.

Vials containing cells were thawed in a 37°C water bath with constant agitation. Cells were washed with DMEM/20% Foetal calf serum (FCS), centrifuged, and a cell count taken using Trypan blue exclusion (Sigma-Aldrich, UK) to determine live cell numbers. The cells were then resuspended in an appropriate pre-warmed medium for use.

Statistics

All results within this study were expressed as the means +/-one standard error. Statistical comparisons were made by the paired t-test, repeated measures ANOVA, or 2-way ANOVA with Bonferroni corrections where appropriate. A value of less than p<0.05 was considered as significant.

Results

MSC characterization

Cells harvested from femoral shaft marrows displayed all the typical characteristics of MSC in culture. At 3 nd passage MSCs were uniformly positive for the mesenchymal markers CD105, CD166, CD44, CD29 but negative for CD45, which is consistent with the known MSC phenotype and excludes contamination of cultures with hematopoietic cells [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF] (figure 1A). Mesenchymal stem cells were induced into adipogenic, osteoblastic and chondrogenic differentiation by culturing MSC, at third passage, in NH Adipodiff medium, NH Osteodiff medium and NH Chondrodiff medium (Miltenyi Biotec, UK) respectively according to the manufacturers' instructions. Adipogenic, osteogenic and chondrogenic differentiation were characterised using the methods described (figure 1B).

Morphology of MSCs after exposure to chemotherapeutic agents

Upon treatment (48h) with cyclophosphamide, or melphalan, significant morphological abnormalities were noted within several treated cultures using phase contrast microscopy (figure 2A). Untreated cultures were morphologically homogenous and characterized by their fibroblast-like appearance with a highly uniform pattern of growth. Cells treated with cyclophosphamide displayed a highly diverse pattern of growth and granular appearance within their cytoplasm. Although having a slightly abnormal appearance, MSC cultures treated with melphalan displayed lower levels of visual morphological changes.

Cytotoxicity assay

Chemotherapy induced MSC cytotoxicity was determined using a colorimetric method for determining the number of viable cells in culture after treatment. Cell viability was calculated by measuring the absorbent value at 490nm based on a significant positive linear correlation between the optical density of the culture media at 490nm and MSC numbers present (data not shown). When examining the chemosensitivity of MSC cultures, the exposure to cyclophosphamide or melphalan did not result in any significant changes in the number of viable cells after treatment for 48 hours at biologically relevant concentrations (p>0.1) (figure 2B).

Expansion capacity of treated MSC cultures

To examine the in-vitro expansion capacity of MSCs treated in-vitro with cyclophosphamide or melphalan, cells were treated and re-plated every 14 days at 7.5x10 4 cells per flask and the total number of cells harvested at the end of each passage was found to calculate the cell population doubling rate (figure 2C). The expansion of untreated marrow MSCs, n=6, was 6.74 population doublings (SE=0.24) after 5 th passage. The expansion of treated MSCs was lower at 5 th passage, with a population doubling of 6.01 for cyclophosphamide (SE=0.52 n=6), and 0.40 population doublings for melphalan (SE=0.40 n=6). Differences in the expansion of untreated and melphalan treated MSCs were statistically significant (p<0.01). No significant differences in expansion were evident when comparing untreated and cyclophosphamide treated MSCs (p>0.05) (figure 2C).

CD44 in-vitro drug sensitivity measurements to chemotherapeutic agents

MSC cultures were treated with chemotherapeutic agents and evaluated for the cell-surface expression of CD44 using flow cytometry, calculated using mean fluorescence intensity (MFI) values. Following incubation for 48 hours with different chemotherapeutic agents, MSCs demonstrated a significant reduction in the CD44 cell membrane surface expression after treatment with cyclophosphamide (p<0.01) (figure 3A). A maximal reduction of 28% in CD44 levels was seen after treatment with cyclophosphamide. No significant changes in MSC CD44 expression were evident after treatment with melphalan (p>0.28) (figure 3A). To examine the longterm effects of chemotherapeutic treatment on CD44 expression by MSC, the levels of CD44 after cyclophosphamide were determined over an 8 week period. Decreased levels of CD44 expression on MSC were evident for up to eight weeks post in-vitro treatment with cyclophosphamide (p<0.05)(figure 3B), with no significant recovery in the level of CD44 expression for the entire culture period post treatment. After eight weeks MSC cultures were terminated due to cell monolayers lifting from the culture flask surface.

Immunoblotting for MSC CD44

Western blot analysis was used to show the expression of CD44 on MSCs, and to confirm its reduction in expression after treatment with cyclophosphamide. CD44 band intensity was measured using ImageQuant™ Version 5.2 software (Molecular Dynamics) and used as a quantitative indicator of protein expression. Detection using a BRIC235 antibody revealed a CD44 protein band at approximately 37kDa for both untreated and treated cell lysates which corresponds to the expected weight of CD44s [START_REF] Cichy | The liberation of CD44[END_REF][START_REF] Ghaffari | CD44 isoforms in normal and leukemic hematopoiesis[END_REF][START_REF] Liu | CD44 and hematologic malignancies[END_REF][START_REF] Ponta | The CD44 protein family[END_REF][START_REF] Sneath | The normal structure and function of CD44 and its role in neoplasia[END_REF] (figure 3C). The CD44 protein band detected by western blot had an estimated weight of 37kDa, which corresponds to the molecular weight of CD44s structure predicted by its amino acid sequence [START_REF] Sneath | The normal structure and function of CD44 and its role in neoplasia[END_REF]. Image analysis using ImageQuant™ software calculated that cyclophosphamide resulted in a significant decrease (p<0.01) in expression of the 37kDa band, with an approximate 22% decrease in CD44 expression. GAPDH expression was used to ensure equal loading of cell lysates.

CD34 + cell transmigration assay

The ability of untreated and cyclophosphamide or melphalan treated MSCs to provide chemotactic signals to hematopoietic progenitor cells and induce transmigration across an endothelial cell barrier were investigated using a HSC migration assay. MSCs cultured within a lower chamber were treated with chemotherapeutic agents, and after 48 hours 6x10 4 CD34 + cells were added to the upper chamber of the transmigration system, with the upper and lower chambers being separated by an endothelial cell layer. The number of hematopoietic progenitor cells that transmigrated through the endothelial barrier of the upper chamber (figure 4A) into the lower chamber after 24 hours was then calculated using a CFU-GM assay (figure 4B). The number of CFU that transmigrated into the lower chamber containing untreated MSCs was 148 (SE = 6). In contrast the numbers of CFU that transmigrated into the lower chamber towards treated MSCs were 106 (SE = 3) (cyclophosphamide), 121 (SE = 6) (melphalan), and 63 (SE = 7) (no MSC).The presence of untreated or treated MSCs within the lower chamber resulted in significantly higher levels of CFU endothelial transmigration into the lower chamber when compared to no MSCs being present (p<0.01). The number of CFU that transmigrated into the lower chamber towards cyclophosphamide or melphalan treated MSCs was significantly lower than the numbers of CFU that transmigrated into the lower chamber towards untreated MSCs (p<0.05). To ensure CD34+ cells were not adhering to the MSC and therefore not being removed for inclusion within the CFU-GM assay, MSC cultures within the lower chamber were trypsinized and put into CFU-GM assay conditions. The presence of CFU-GM were absent from the adherent cell populations obtained (data not shown).

Hematopoietic colony-forming assay

The ability of MSC-derived stromal cells to support hematopoiesis was compared post cyclophosphamide or melphalan treatment in long term hematopoietic cell cultures. MSCs from all groups in long-term culture conditions developed the same microscopic appearance as stroma derived from fresh marrow buffy coat samples as reported by Wexler et al [START_REF] Wexler | Adult bone marrow is a rich source of human mesenchymal 'stem' cells but umbilical cord and mobilized adult blood are not[END_REF]. All adherent MSC-derived stromal cultures were primarily seeded with hematopoietic CD34 + cells derived from the same cord blood source. Co-culture of MSCs with allogeneic cord blood CD34positive hematopoietic progenitor cells resulted in the formation of cobblestone areas representative of hematopoietic progenitor cell proliferation and differentiation (figure 5). Assessing hematopoietic activity, using a 2-way ANOVA with Bonferroni corrections, tests indicated that over the 6 week culture period there was a significant reduction in the numbers of CFU-GM present for up to 3 weeks in cultures seeded onto cyclophosphamide and melphalan treated MSC derived stromal cultures, when compared to the numbers of CFU-GM supported by untreated MSC derived stromal cultures, (p<0.05), (figure 5).

Discussion

Chemotherapy and stem cell transplantation are well established treatments of hematological disorders and other cancers. The adverse effects of the chemotherapeutic agents used in these treatments on HSCs are well known, however the effects on the MSCs residing in the bone marrow are less well characterized. Studies have demonstrated that a recipient's stromal cells are damaged after bone marrow transplantation [START_REF] Domenech | Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation[END_REF][START_REF] Galotto | Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients[END_REF][START_REF] O'flaherty | Bone marrow stromal function from patients after bone marrow transplantation[END_REF], or even from chemotherapeutic drugs alone [START_REF] Domenech | Changes in the functional capacity of marrow stromal cells after autologous bone marrow transplantation[END_REF][START_REF] Galotto | Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients[END_REF][START_REF] O'flaherty | Bone marrow stromal function from patients after bone marrow transplantation[END_REF][START_REF] Tabilio | Effect of chemotherapy for acute myelogenous leukemia on hematopoietic and fibroblast marrow progenitors[END_REF][START_REF] Corazza | Bone marrow stroma damage induced by chemotherapy for acute lymphoblastic leukemia in children[END_REF][START_REF] Banfi | High-dose chemotherapy shows a dose-dependent toxicity to bone marrow osteoprogenitors: a mechanism for post-bone marrow transplantation osteopenia[END_REF][START_REF] Cohen | Effect of chemotherapy and irradiation on interactions between stromal and hemopoietic cells in vitro[END_REF][START_REF] Domaratskaia | Alkylating damage by dipin of hematopoietic and stromal cells of the bone marrow[END_REF][START_REF] Domenech | Haemopoiesis of transplanted patients with autologous marrows assessed by long-term marrow culture[END_REF][START_REF] Fried | Effects of radiation on hematopoietic stroma[END_REF]. We have recently demonstrated a significant reduction in MSC expansion and MSC CD44 expression by MSCs derived from patients receiving HDC regimens for hematological malignancies [START_REF] Kemp | Chemotherapyinduced mesenchymal stem cell damage in patients with hematological malignancy[END_REF]. Examining the effects of chemotherapeutic agents on patient MSC in vivo is difficult due to variability in patients and differences in the drug combinations used, both of which could have implications on MSC function. With drugs not being commonly used as single agents during HDC regimens there is a lack of data comparing the short or long term effects these drugs have on patients post treatment. To address these problems the effects of the commonly used alkylating chemotherapeutic agents cyclophosphamide and melphalan on human bone marrow MSC were evaluated in-vitro.

Both cyclophosphamide and melphalan belong to a class of alkylating agents known as nitrogen mustard derivatives. They are widely used in high-dose chemotherapeutic treatment of malignant disorders and in the preparative bone-marrow conditioning regimens pre-stem cell transplant. These compounds function as chemotherapeutic agents by the formation of DNA alkyl adducts and inter-strand DNA-cross links, inducing cellular apoptosis [START_REF] Zheng | Repair of DNA interstrand cross-links: interactions between homology-dependent and homology-independent pathways[END_REF]. Knowledge of the clinical pharmacokinetics and pharmacodynamics of these agents administered in high doses is critical for the safe and efficient use of these regimens [START_REF] Huitema | The clinical pharmacology of alkylating agents in high-dose chemotherapy[END_REF]. The concentration of both cyclophosphamide or melphalan within the bone marrow of patients undergoing high dose chemotherapy will be variable even after doses are adjusted for body surface area or weight, as there is considerable pharmacokinetic variability among patients receiving chemotherapy [START_REF] Davies | In vitro effects of chemotherapeutic agents on human osteoblast-like cells[END_REF]. The drug concentrations used to treat MSCs in this study were therefore based on the mean peak concentrations of either cyclophosphamide or melphalan observed in the plasma of patients undergoing high-dose chemotherapy treatments [START_REF] Chen | Nonlinear pharmacokinetics of cyclophosphamide in patients with metastatic breast cancer receiving high-dose chemotherapy followed by autologous bone marrow transplantation[END_REF][START_REF] Li | Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents[END_REF][START_REF] Lazarus | Intensive melphalan chemotherapy and cryopreserved autologous bone marrow transplantation for the treatment of refractory cancer[END_REF][START_REF] Pinguet | Pharmacokinetics of high-dose intravenous melphalan in patients undergoing peripheral blood hematopoietic progenitor-cell transplantation[END_REF][START_REF] Alberts | Comparative pharmacokinetics of chlorambucil and melphalan in man[END_REF].

Primarily the sensitivity of bone marrow MSCs to cyclophosphamide or melphalan induced cell death was investigated. Results indicated that exposure to these drugs did not cause any significant level of cell death to MSC cultures after 48 hours at the drug concentrations used. This resistance to apoptosis by cyclophosphamide is comparable to results seen by Li et al at identical drug concentrations [START_REF] Li | Differential damage and recovery of human mesenchymal stem cells after exposure to chemotherapeutic agents[END_REF], and may partially explain why MSCs can be harvested from bone-marrow derived from patients who have received prior high dose chemotherapy [START_REF] Kemp | Chemotherapyinduced mesenchymal stem cell damage in patients with hematological malignancy[END_REF][START_REF] Zhao | Assessment of bone marrow mesenchymal stem cell biological characteristics and support hemotopoiesis function in patients with chronic myeloid leukemia[END_REF]. Although no significant loss of cell viability was observed, when investigating the expansion potential of MSCs post treatment, the mean expansion of untreated MSCs was over five times that seen of MSCs treated with melphalan after five passages. In contrast, cyclophosphamide treatment did not result in any reduction in MSC expansion. The cytotoxic effects of chemotherapeutic drugs, that result in DNA adducts, will result in alterations in gene expression and induction of mechanisms by which cells can detect DNA damage and control DNA repair. The DNA damage response, involving sensory and effector proteins, is strongly tied with cellular proliferation, cell cycle arrest, cellular senescence and apoptosis [START_REF] Davies | In vitro effects of chemotherapeutic agents on human osteoblast-like cells[END_REF][START_REF] Hochhauser | Modulation of chemosensitivity through altered expression of cell cycle regulatory genes in cancer[END_REF]. Although investigating the exact mechanisms by which melphalan causes a decrease in MSC expansion would be desirable, it does demonstrate that chemotherapy disrupts the replicating ability of MSC, and corresponds with reductions in proliferative capacity of MSC harvested from patients who had received prior chemotherapy treatment [START_REF] Kemp | Chemotherapyinduced mesenchymal stem cell damage in patients with hematological malignancy[END_REF]. This chemotherapy induced defect on proliferation is therefore a great concern as the ability/capacity for extensive proliferation and self-renewal is an essential property of a stem cell population. It could also explain why low numbers of mesenchymal progenitors are evident in patients after chemotherapy exposure [START_REF] Galotto | Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients[END_REF][START_REF] Banfi | High-dose chemotherapy shows a dose-dependent toxicity to bone marrow osteoprogenitors: a mechanism for post-bone marrow transplantation osteopenia[END_REF][START_REF] Domaratskaia | Alkylating damage by dipin of hematopoietic and stromal cells of the bone marrow[END_REF].

MSCs have distinct structural properties from those of fully differentiated cells. Following exposure of MSC cultures to various chemotherapeutic agents structural abnormalities were evident with disruptions to the cell membrane morphology. MSC membrane surface expression of CD44 was also studied in response to the exposure of chemotherapeutic agents in-vitro as low CD44 levels seen on patients MSC after receiving high dose chemotherapy for hematological disorders [START_REF] Kemp | Chemotherapyinduced mesenchymal stem cell damage in patients with hematological malignancy[END_REF]. CD44 is an adhesion molecule that plays a critical role in normal hematopoiesis [START_REF] Khaldoyanidi | Correlation between nicotine-induced inhibition of hematopoiesis and decreased CD44 expression on bone marrow stromal cells[END_REF][START_REF] Moll | Two different functions for CD44 proteins in human myelopoiesis[END_REF], and it is the stromal microenvironment consisting of stromal cells and extracellular matrix that is thought to regulate and support the future fate of stem cells and committed progenitors along specific lineages [START_REF] Majumdar | Human marrowderived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages[END_REF][START_REF] Majumdar | Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells[END_REF][START_REF] Reese | Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction[END_REF]. Using both flow cytometry and western blot techniques, the exposure of MSC cultures to treatment with cyclophosphamide resulted in a decrease in MSC CD44 expression, with a maximal decrease of 28%. Decreased levels of CD44 expression on MSC were evident for prolonged periods post treatment showing no sign of recovery.

The homing and engraftment of transplanted HSCs to the bone-marrow stem cell niche is essential for the establishment of intact hematopoiesis after high dose chemotherapy and/or total body irradiation in conjunction with stem cell transplantation. It is thought that normal cell migratory processes involve events mediated through multiple adhesion/chemokine ligand interactions. Bone marrow cells are thought to secrete chemoattractants which facilitate the homing of circulating stem cells to the affected tissue to promote organ regeneration or tissue repair. The ability of MSCs to provide support for HSC migration after exposure to different chemotherapeutic drugs used in conditioning regimens in largely unknown. Intensive bone marrow transplant conditioning regimens, involving exposure to high concentrations of either cyclophosphamide or melphalan, create space within the marrow by removing the host's hematopoietic cells and also malignant cells in disease states in which the bone marrow is affected. In addition, chemotherapy agents used can also induce changes in vivo known to increase the homing potential of HSCs through disruption of the bone marrow endothelium barrier [START_REF] Szumilas | Effect of stem cell mobilization with cyclophosphamide plus granulocyte colony-stimulating factor on morphology of haematopoietic organs in mice[END_REF], and increased secretion of cytokines and chemokines which have impacts on HSC migration and repopulation [START_REF] Cottler-Fox | Stem cell mobilization[END_REF][START_REF] Lapidot | How do stem cells find their way home?[END_REF][START_REF] Ponomaryov | Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function[END_REF][START_REF] Zhao | Soluble factor(s) from bone marrow cells can rescue lethally irradiated mice by protecting endogenous hematopoietic stem cells[END_REF][START_REF] Lapidot | Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells[END_REF].

The in-vitro transmigration model used in this study allows analysis of trans-endothelial migration of hematopoietic progenitor cells towards untreated and chemotherapeutically treated MSC layers. Homing is a fairly rapid process occurring no longer than 1-2 days [START_REF] Lapidot | How do stem cells find their way home?[END_REF], thus transmigration was observed during a period of 24 hours in these experiments. Firstly, it was demonstrated that the presence of MSCs (treated or untreated) within the lower chamber of the migration assay set-up resulted in an increased trans-endothelial migration of hematopoietic progenitors into the lower chamber when compared to no MSCs being present. This provided evidence that both treated and untreated MSC monolayers were able to produce chemo-attractive signals to assist in trans-endothelial passage and facilitate hematopoietic progenitor cell homing into the lower chamber.

Subsequently it was found that the numbers of hematopoietic progenitors migrating through the endothelial layer, towards the MSCs within the lower chamber over a 24 hour period, were reduced after exposure of the MSCs to either cyclophosphamide or melphalan. As only MSCs were exposed to the chemotherapeutic drugs, the reduction in the rate of hematopoietic cell migration must result from a change in the secretion of a single or variety of soluble factors by MSCs post chemotherapeutic insult. These changes in the expression of soluble factors reducing HSC migration are either acting indirectly on the endothelial cells effecting trans-endothelial passage, or acting directly on the HSCs affecting their migration potential. Previous data has shown that there is increased homing of transplanted progenitor cells in non-irradiated non-ablative mice compared to TBI ablative preconditioned mice [START_REF] Collis | Hematopoietic progenitor stem cell homing in mice lethally irradiated with ionizing radiation at differing dose rates[END_REF][START_REF] Hendrikx | Homing of fluorescently labeled murine hematopoietic stem cells[END_REF]. This inhibitory effect on stem cell homing caused chemotherapy exposure to MSC could in part explain the decrease in stem cell homing after ablative conditioning regimens.

Engraftment leading to hematopoietic recovery is vital following intensive chemotherapy/radiotherapy preparative regimens and stem cell transplantation. This engraftment is initiated by differentiating hematopoietic progenitor cells supported by the bone-marrow microenvironment. Engraftment may be slow or even fail following transplantation leading to prolonged pancytopenia or even death. This can happen despite an intensive preparative regimen, an adequate stem cell dose and complete donor chimerism [START_REF] Bacigalupo | Mesenchymal stem cells and haematopoietic stem cell transplantation[END_REF]. It has been suggested that this lack of engraftment is due to functional damage caused by chemotherapeutic agents to the marrow microenvironment resulting in an inability to support the expansion of infused HSCs [START_REF] Galotto | Stromal damage as consequence of high-dose chemo/radiotherapy in bone marrow transplant recipients[END_REF][START_REF] Tabilio | Effect of chemotherapy for acute myelogenous leukemia on hematopoietic and fibroblast marrow progenitors[END_REF]. Whether chemotherapy exposure affects MSC ability to form functional stroma and/or support hematopoiesis within the bone-marrow and has a role in changing hematopoietic engraftment kinetics in transplant patients is unknown.

In long-term hematopoietic cell cultures the CFU-GM assay is used as an indirect measure of the number of primitive hematopoietic cells engrafting on MSC-derived stromal cells. Thus, the CFU-GM readout may be used to assess the ability of MSC-derived stromal cells to support hematopoiesis [START_REF] Dexter | Conditions controlling the proliferation of haemopoietic stem cells in vitro[END_REF][START_REF] De Wynter | Assays for the assessment of human hematopoietic stem cells[END_REF]. Data in this study demonstrate that MSC-derived stromal cells treated with either cyclophosphamide or melphalan display an abnormal stromal function in supporting early hematopoietic progenitor cells, with a reduced progenitor cell support for up to three weeks post treatment in long-term cultures. The role of MSC damage in engraftment kinetics is unknown; however it is known that MSCs can produce of a number of early-acting cytokines which maintain HSCs in quiescence or promote their self-renewal, and also a variety of interleukins and cytokines which act on more mature hematopoietic progenitors [START_REF] Majumdar | Human marrowderived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages[END_REF][START_REF] Majumdar | Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells[END_REF]. Successful hematopoietic engraftment is heralded in 2-3 weeks by the appearance of donor progenitor cells in the circulation of the recipient [START_REF] Riley | Hematologic aspects of myeloablative therapy and bone marrow transplantation[END_REF]. The reduction in hematopoietic support by MSC-derived stroma observed within this time period, after cyclophosphamide or melphalan treatment, could therefore have large influences on patient engraftment and recovery.

Conclusion

Disruptions in the ability of MSCs to undertake their normal biological function caused by cytotoxic damage during chemotherapy treatment could have huge consequences on hematopoiesis and tissue formation in vivo, as the multi-potential ability of MSC allows them to maintain host tissues during normal life and participate in tissue regeneration or repair in response to disease or injury [START_REF] Javazon | Mesenchymal stem cells: paradoxes of passaging[END_REF]. Within this study the exposure of MSCs to the chemotherapeutic agents cyclophosphamide or melphalan had strong negative effects on MSC expansion and CD44 expression. In addition, changes were seen in the ability of MSCs to support hematopoietic cell migration and repopulation. Differences in cyclophosphamide or melphalan treatment on MSC function were evident.

However, due to these drugs not being commonly used as single agents during high dose chemotherapy regimens there is a lack of data comparing the short or long term effects these drugs have on patients post treatment.

Collectively, these observations implicate the potential disadvantages in the use of autologous MSCs in chemotherapeutically pre-treated patients for future therapeutic strategies. Furthermore, this study lends to the hypothesis that if damage caused by chemotherapeutic agents to marrow MSCs is substantial, it would be logical to use cultured allogeneic MSCs therapeutically to assist or repair the marrow microenvironment after HDC.

Figures and

Figure 1 .

 1 Figure 1. (A) The phenotype of cells within MSC culture at 3rd passage (n=6). Results are expressed as the percentage of cells expressing the phenotypic cellular markers CD105, CD166, CD29, CD44 and CD45 in MSC culture (+/-SEM). (B) Images depicting MSC cultures at 3rd passage differentiated down the adipogenic, osteogenic and chondrogenic lineages. Adipogenic differentiation was visualized by the accumulation of lipidcontaining vacuoles which stain red with oil red O (i). Osteogenic differentiation was visualized by the presence of calcium (ii). Chondrogenic differentiation was characterized by the immunofluorescent detection of aggrecan (green)/ nuclei (blue) (iii). Undifferentiated MSC cultures at 3 rd passage show absence of staining with oil red O (iv), Alizarin red (v) and aggrecan (vi).

Figure 2 .

 2 Figure 2. (A) Representative phase contrast images of MSC cultures treated with cyclophosphamide or melphalan. Cells treated with chemotherapeutic agents displayed a mixed morphology in culture with high levels of granularity present within their cytoplasm. (B) MSC viability after chemotherapeutic treatment. Results are expressed as a mean +/-SEM after treatment of MSC cultures with cyclophosphamide (n=6) or melphalan (n=6) (no significant differences in cell viability were evident between control and treated cultures, p>0.05). (C) Melphalan treatment damages the in-vitro long-term expansion capacity of MSCs. The graph depicts the expansion of untreated MSCs (n=6), or MSCs treated with the chemotherapeutics cyclophosphamide (n=6) or melphalan (n=6). Results are expressed as the mean +/-(SEM) (untreated vs melphalan p<0.01; untreated vs cyclophosphamide p>0.05; ANOVA).

Figure 3 .

 3 Figure 3. Cyclophosphamide causes a significant reduction in MSC-CD44 expression. (A) The levels of CD44 expression, using MFI values, on MSCs exposed to cyclophosphamide or melphalan for 48 hours. Untreated (n=6), cyclophosphamide (n=6) and melphalan (n=6). (B) The levels of CD44 expression, using MFI values, on MSCs exposed to cyclophosphamide over an 8 week period post exposure. Results are expressed as the mean MFI level (+/-SEM) (untreated vs cyclophosphamide; *p<0.05). (C) Immunoblotting of CD44 on MSC samples post exposure to cyclophosphamide for 48 hours (n=4). Upper panels correspond to CD44; lower panel corresponds to GAPDH which was used to ensure equal protein loading.

Figure 4 .

 4 Figure 4. The chemotactic property of MSCs is decreased after exposure to chemotherapeutic agents. (A) Phase contrast images of human umbilical vein endothelial cells in culture prior to being used in the CD34 + cell transmigration assay. Images taken at (i) low and (ii) high magnification. (B) The frequency of hematopoietic progenitor cells (CFU-GM) transmigrating an endothelial cell layer towards an untreated MSC layer (Untreated), a chemotherapeutically treated MSC layer (Cyclophosphamide or Melphalan) or no MSC layer (No MSC). The number of transmigrated cells was measured after 24 hours. Results are expressed as the mean +/-SEM (*p<0.05 when compared with number of CFU-GM values in the untreated control group).

Figure 5 .

 5 Figure 5. (A) MSC-derived stroma treated with melphalan or cyclophosphamide display an abnormal stromal function. The graph depicts the number of CFU-GM present in culture after seeding cord blood CD34 + cells into co-culture with MSC-derived stromal long term cultures exposed to cyclophosphamide or melphalan (n =6). Results are expressed as the mean (+/-SEM). (untreated vs cyclophosphamide or melphalan p<0.05 (3wks); ANOVA). (B) A phase contrast photomicrograph showing confluent and adherent MSC-derived stromal cells (white arrows) supporting the growth and differentiation of non-adherent cord blood derived CD34 + cells (black arrows) when seeded into long-term co-culture.

Acknowledgements

We wish to express our gratitude to the doctors and nurses at the Southmead Hospital, Bristol, for bone marrow collections; and to all patients who donated marrow samples. We would also like to thank midwives at the Central Delivery Suite, Southmead Hospital, Bristol, for cord blood collections; and mothers who donated cord blood. This work was supported by a PhD bursary from the University of the West of England, and also in part by the funding provided by the Transplant Trust.