Initial mycophenolate dose in tacrolimus treated renal transplant recipients, a cohort study comparing leukopaenia, rejection and long-term graft function.

The evidence supporting an initial mycophenolate mofetil (MMF) dose of 2 g daily in tacrolimus-treated renal transplant recipients is limited. In a non-contemporaneous single-centre cohort study we compared the incidence of leukopaenia, rejection and graft dysfunction in patients initiated on MMF 1.5 g and 2 g daily. Baseline characteristics and tacrolimus trough levels were similar by MMF group. MMF doses became equivalent between groups by 12-months post-transplant, driven by dose reductions in the 2 g group. Leukopaenia occurred in 42.4% of patients by 12-months post-transplant. MMF 2 g was associated with a 1.80-fold increased risk of leukopaenia compared to 1.5 g. Rejection occurred in 44.8% of patients by 12-months post-transplantation. MMF 2 g was associated with half the risk of rejection relative to MMF 1.5 g. Over the first 7-years post-transplantation there was no difference in renal function between groups. Additionally, the development of leukopaenia or rejection did not result in reduced renal function at 7-years post-transplant. Leukopaenia was not associated with an increased incidence of serious infections or rejection. This study demonstrates the initial MMF dose has implications for the incidence of leukopaenia and rejection. Since neither dose produced superior long-term graft function, clinical equipoise remains regarding the optimal initial mycophenolate dose in tacrolimus-treated renal transplant recipients.

Phase 1 trial of vorolanib (CM082) in combination with everolimus in patients with advanced clear-cell renal cell carcinoma.

BACKGROUND: Vorolanib (X-82, CM082) is a multi-target tyrosine kinase inhibitor. This study aimed to evaluate the tolerability, safety, pharmacokinetics and antitumor activities of vorolanib plus everolimus (an inhibitor of mammalian target of rapamycin). METHODS: Patients had histologically or cytologically confirmed advanced RCC and failed with standard therapy were eligible for this study. Dose-escalated combinations of vorolanib (100, 150 or 200 mg once daily) with everolimus (5 mg once daily) were administered on 28-day cycles until disease progression or unacceptable toxicity using a conventional 3 + 3 dose-escalation design. FINDINGS: 22 patients (100 mg n = 4, 150 mg n = 3, 200 mg n = 15) were enrolled. Only one patient experienced dose-limiting toxicity (DLT, grade 4 thrombocytopenia) in the vorolanib 200 mg combination cohort, and the maximum tolerated dose (MTD) was not reached. The most common treatment-related adverse events were proteinuria (100%), leukopenia (77%), hypercholesterolaemia (77%), increased low-density lipoprotein (68%), hypertriglyceridaemia (64%), hyperglycaemia (59%), and fatigue (55%). Most treatment-related adverse events were grade 1 to 2, with grade 3 or higher toxicities mostly seen in the 200 mg cohort. Single dosing of vorolanib demonstrated dose-proportional increases in the Cmax and AUC, and observed short t1/2z ranging from 4.74±1.44 to 12.89±7.49 h. The pharmacokinetic parameters for everolimus were similar among all cohorts. Of 19 evaluable patients, the ORR and DCR was 32% (n = 6, 95% CI, 13-57%) and 100% (95% CI, 82-100%), respectively. INTERPRETATION: Combination therapy of vorolanib 200 mg plus everolimus 5 mg once daily is potentially effective with potential activity. Further evaluation of the combination in advanced RCC patients is ongoing (NCT03095040). FUNDING: Betta Pharmaceutical Co., Ltd., Hangzhou, China.

Reticular dysgenesis: international survey on clinical presentation, transplantation, and outcome.

Reticular dysgenesis (RD) is a rare congenital disorder defined clinically by the combination of severe combined immunodeficiency (SCID), agranulocytosis, and sensorineural deafness. Mutations in the gene encoding adenylate kinase 2 were identified to cause the disorder. Hematopoietic stem cell transplantation (HSCT) is the only option to cure this otherwise fatal disease. Retrospective data on clinical presentation, genetics, and outcome of HSCT were collected from centers in Europe, Asia, and North America for a total of 32 patients born between 1982 and 2011. Age at presentation was <4 weeks in 30 of 32 patients (94%). Grafts originated from mismatched family donors in 17 patients (55%), from matched family donors in 6 patients (19%), and from unrelated marrow or umbilical cord blood donors in 8 patients (26%). Thirteen patients received secondary or tertiary transplants. After transplantation, 21 of 31 patients were reported alive at a mean follow-up of 7.9 years (range: 0.6-23.6 years). All patients who died beyond 6 months after HSCT had persistent or recurrent agranulocytosis due to failure of donor myeloid engraftment. In the absence of conditioning, HSCT was ineffective to overcome agranulocytosis, and inclusion of myeloablative components in the conditioning regimens was required to achieve stable lymphomyeloid engraftment. In comparison with other SCID entities, considerable differences were noted regarding age at presentation, onset, and type of infectious complications, as well as the requirement of conditioning prior to HSCT. Although long-term survival is possible in the presence of mixed chimerism, high-level donor myeloid engraftment should be targeted to avoid posttransplant neutropenia.

NUDT15 R139C causes thiopurine-induced early severe hair loss and leukopenia in Japanese patients with IBD.

The efficacy of thiopurines, including azathioprine (AZA) and 6-mercaptopurine (6MP), has been demonstrated for the treatment of inflammatory bowel disease (IBD). The most common and serious adverse event of treatment with thiopurines altered by doctors is leukopenia. Hair loss is also a serious event that could be a critical reason for patients to decline thiopurine treatment. Thiopurine-induced severe hair loss causes cosmetic problems, and it takes a long time to recover. In a recent study, NUDT15 R139C was strongly associated with thiopurine-induced leukopenia in Korean and Caucasian populations. In this study, we performed an association study to investigate and replicate the association of R139C with adverse events of thiopurines in Japanese patients. A total of 142 Japanese patients with IBD, with histories of thiopurine treatment, were examined. NUDT15 R139C was genotyped using a custom TaqMan genotyping assay. Adverse events including leukopenia were reviewed from medical records. The 6MP dose was adjusted to AZA equivalents by multiplying with 2 as a thiopurine dose. Five patients developed severe hair loss and all of them were risk homozygous (T/T) for R139C. No early severe hair loss was observed in patients with the C/T or C/C genotype (P=3.82 × 10(-16), odds ratio=212). The association of R139C with early (<8 weeks) leukopenia (white blood cells<3000 mm(-3)), which was previously reported in Korean patients, was replicated in our Japanese IBD cohort (P=1.92 × 10(-16), odds ratio=28.4). However, we could not confirm the association with late leukopenia in the Japanese subjects. Patients with the C/T genotype discontinued treatment or required thiopurine dose reduction significantly earlier than patients with the C/C genotype (P=1.45 × 10(-4)); however, on manipulating the doses, there was no significant difference in the thiopurine continuation rates between the groups. In the maintenance period, the frequencies of 6MP usage were higher, and the doses of thiopurines were significantly lower in patients with the C/T genotype than in those with the C/C genotype (0.574±0.316 mg kg(-1) per day vs 1.03±0.425 mg kg(-1) per day, P=6.21 × 10(-4)). NUDT R139C was significantly associated with early severe hair loss in Japanese patients with IBD. We also verified the previously reported association of R139C with early leukopenia in a different East Asian population. It is recommended that treatment with thiopurines should be avoided for patients with the T/T genotype. Low-dose 6MP (0.2-0.3 mg kg(-1) per day) could be used rather than AZA for the patients with C/T genotype to continue thiopurine treatments. However, late leukopenia and other several adverse events could not be completely predicted by R139C genotypes.

AK2 deficiency compromises the mitochondrial energy metabolism required for differentiation of human neutrophil and lymphoid lineages.

Reticular dysgenesis is a human severe combined immunodeficiency that is primarily characterized by profound neutropenia and lymphopenia. The condition is caused by mutations in the adenylate kinase 2 (AK2) gene, resulting in the loss of mitochondrial AK2 protein expression. AK2 regulates the homeostasis of mitochondrial adenine nucleotides (ADP, ATP and AMP) by catalyzing the transfer of high-energy phosphate. Our present results demonstrate that AK2-knocked-down progenitor cells have poor proliferative and survival capacities and are blocked in their differentiation toward lymphoid and granulocyte lineages. We also observed that AK2 deficiency impaired mitochondrial function in general and oxidative phosphorylation in particular - showing that AK2 is critical in the control of energy metabolism. Loss of AK2 disrupts this regulation and leads to a profound block in lymphoid and myeloid cell differentiation.

Reticular dysgenesis-associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

Susceptibility to 6-MP toxicity conferred by a NUDT15 variant in Japanese children with acute lymphoblastic leukaemia.

Genotyping of TPMT prior to 6-mercaptopurine (6-MP) administration in acute lymphoblastic leukaemia (ALL) patients has been integrated into clinical practice in some populations of European ancestry. However, the comparable rates of 6-MP myelotoxicity, but rarity of TPMT variants, in Asians suggest that major determinants have yet to be discovered in this population. We genotyped 92 Japanese paediatric ALL patients for NUDT15 rs116855232, a 6-MP toxicity-related locus discovered in Asians. Logistic regression and survival analysis were used to evaluate its association with leucopenia, hepatotoxicity, 6-MP dose reduction, therapy interruption and event-free survival. The allele frequency of rs116855232 was 0·16, and leucopenia was more common in carriers of the T allele (odds ratio, 7·20; 95% confidence interval, 2·49-20·80; P = 2·7 × 10(-4) ). As leucopenia results in 6-MP dose reduction, we observed average doses during maintenance therapy of 40·7, 29·3 and 8·8 mg/m(2) for patients with CC, CT and TT genotypes, respectively (P < 0·001). Hepatotoxicity was observed only in CC genotype patients. Event-free survival did not significantly differ by NUDT15 genotype. rs116855232 is an important determinant of 6-MP myelotoxicity in Japanese children with ALL and may represent the most robust toxicity-related locus in Asians to date. Considerations for clinical application may be warranted.

Carboxypeptidase B2 deficiency reveals opposite effects of complement C3a and C5a in a murine polymicrobial sepsis model.

BACKGROUND AND OBJECTIVES: Carboxypeptidase B2 (CPB2) is a basic carboxypeptidase with fibrin and complement C3a and C5a as physiological substrates. We hypothesized that in polymicrobial sepsis, CPB2-deficient mice would have sustained C5a activity, leading to disease exacerbation. METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture (CLP). RESULTS: Contrary to our hypothesis, Cpb2(-/-) mice had significantly improved survival, with reduced lung edema, less liver and kidney damage, and less disseminated intravascular coagulation. Hepatic pro-CPB2 was induced by CLP, leading to increased pro-CPB2 levels. Thrombomodulin present on mesothelium supported thrombin activation of pro-CPB2. Both wild-type and Cpb2(-/-) animals treated with a C5a receptor antagonist had improved survival, demonstrating that C5a was detrimental in this model. Treatment with a fibrinolysis inhibitor, tranexamic acid, caused a decrease in survival in both genotypes; however, the Cpb2(-/-) animals retained their survival advantage. Administration of a C3a receptor antagonist exacerbated the disease in both wild-type and Cpb2(-/-) mice and eliminated the survival advantage of Cpb2(-/-) mice. C5a receptor is expressed in both peritoneal macrophages and neutrophils; in contrast, C3a receptor expression is restricted to peritoneal macrophages, and C3a induced signaling in macrophages but not neutrophils. CONCLUSIONS: While C5a exacerbates the peritonitis, resulting in a deleterious generalized inflammatory state, C3a activation of peritoneal macrophages may limit the initial infection following CLP, thereby playing a diametrically opposing protective role in this polymicrobial sepsis model.

A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia.

Thiopurine therapy, commonly used in autoimmune conditions, can be complicated by life-threatening leukopenia. This leukopenia is associated with genetic variation in TPMT (encoding thiopurine S-methyltransferase). Despite a lower frequency of TPMT mutations in Asians, the incidence of thiopurine-induced leukopenia is higher in Asians than in individuals of European descent. Here we performed an Immunochip-based 2-stage association study in 978 Korean subjects with Crohn's disease treated with thiopurines. We identified a nonsynonymous SNP in NUDT15 (encoding p.Arg139Cys) that was strongly associated with thiopurine-induced early leukopenia (odds ratio (OR) = 35.6; P(combined) = 4.88 × 10(-94)). In Koreans, this variant demonstrated sensitivity and specificity of 89.4% and 93.2%, respectively, for thiopurine-induced early leukopenia (in comparison to 12.1% and 97.6% for TPMT variants). Although rare, this SNP was also strongly associated with thiopurine-induced leukopenia in subjects with inflammatory bowel disease of European descent (OR = 9.50; P = 4.64 × 10(-4)). Thus, NUDT15 is a pharmacogenetic determinant for thiopurine-induced leukopenia in diverse populations.

Differential expression of adenine nucleotide converting enzymes in mitochondrial intermembrane space: a potential role of adenylate kinase isozyme 2 in neutrophil differentiation.

Adenine nucleotide dynamics in the mitochondrial intermembrane space (IMS) play a key role in oxidative phosphorylation. In a previous study, Drosophila adenylate kinase isozyme 2 (Dak2) knockout was reported to cause developmental lethality at the larval stage in Drosophila melanogaster. In addition, two other studies reported that AK2 is a responsible gene for reticular dysgenesis (RD), a human disease that is characterized by severe combined immunodeficiency and deafness. Therefore, mitochondrial AK2 may play an important role in hematopoietic differentiation and ontogenesis. Three additional adenine nucleotide metabolizing enzymes, including mitochondrial creatine kinases (CKMT1 and CKMT2) and nucleoside diphosphate kinase isoform D (NDPK-D), have been found in IMS. Although these kinases generate ADP for ATP synthesis, their involvement in RD remains unclear and still an open question. In this study, mRNA and protein expressions of these mitochondrial kinases were firstly examined in mouse ES cells, day 8 embryos, and 7-week-old adult mice. It was found that their expressions are spatiotemporally regulated, and Ak2 is exclusively expressed in bone marrow, which is a major hematopoietic tissue in adults. In subsequent experiments, we identified increased expression of both AK2 and CKMT1 during macrophage differentiation and exclusive production of AK2 during neutrophil differentiation using HL-60 cells as an in vitro model of hematopoietic differentiation. Furthermore, AK2 knockdown specifically inhibited neutrophil differentiation without affecting macrophage differentiation. These data suggest that AK2 is indispensable for neutrophil differentiation and indicate a possible causative link between AK2 deficiency and neutropenia in RD.

Adenylate kinase 2 links mitochondrial energy metabolism to the induction of the unfolded protein response.

The unfolded protein response (UPR) is a homeostatic signaling mechanism that balances the protein folding capacity of the endoplasmic reticulum (ER) with the secretory protein load of the cell. ER protein folding capacity is dependent on the abundance of chaperones, which is increased in response to UPR signaling, and on a sufficient ATP supply for their activity. An essential branch of the UPR entails the splicing of XBP1 mRNA to form the XBP1 transcription factor. XBP1 has been shown to be required during adipocyte differentiation, enabling mature adipocytes to secrete adiponectin, and during differentiation of B cells into antibody-secreting plasma cells. Here we find that adenylate kinase 2 (AK2), a mitochondrial enzyme that regulates adenine nucleotide interconversion within the intermembrane space, is markedly induced during adipocyte and B cell differentiation. Depletion of AK2 by RNAi impairs adiponectin secretion in 3T3-L1 adipocytes, IgM secretion in BCL1 cells, and the induction of the UPR during differentiation of both cell types. These results reveal a new mechanism by which mitochondria support ER function and suggest that specific mitochondrial defects may give rise to impaired UPR signaling. The requirement for AK2 for UPR induction may explain the pathogenesis of the profound hematopoietic defects of reticular dysgenesis, a disease associated with mutations of the AK2 gene in humans.

Assessment of thiopurine methyltransferase activity in patients prescribed azathioprine or other thiopurine-based drugs.

OBJECTIVES: To examine whether pretreatment determination of thiopurine methyltransferase (TPMT) enzymatic activity (phenotyping) or TPMT genotype, to guide thiopurine therapy in chronic autoimmune disease patients, reduces treatment harms. Other objectives included assessing: preanalytic, analytic, and postanalytic requirements for TPMT testing; diagnostic accuracy of TPMT genotyping versus phenotyping; association of thiopurine toxicity with TPMT genotypic or phenotypic status; and costs of testing, care, and treating drug-associated complications. DATA SOURCES: MEDLINE®, EMBASE®, and Healthstar were searched from inception to May 2010; the Cochrane Library® to October 2009; and BIOSIS®, Genetics Abstracts, and EconLit™ to May 2009, for English language records. REVIEW METHODS: A reviewer screened records, and a second reviewer verified exclusions and subsequent selection of relevant studies. Studies in patients with leukemia and organ transplant were excluded. Additionally, laboratories that provide TPMT analytical services were surveyed to assess means of TPMT testing in practice. Where possible, risk of bias was assessed using standard criteria. Meta-analyses estimated diagnostic sensitivity, and specificity; and odds ratios of associations. RESULTS: 1790 titles or abstracts, and 538 full text records were screened. 114 observational studies and one RCT were included. Majority of studies were rated fair quality, except for diagnostic studies with 37 percent of studies rated poor. In general, there were few patients who were homozygous (or compound heterozygous) for TPMT variant alleles in the included studies limiting applicability. There is insufficient evidence examining effectiveness of pretesting in terms of reduction in clinical adverse events. Sufficient preanalytical data were available regarding preferred specimen collection, stability and storage conditions for TPMT testing. There was no clinically significant effect of age, gender, various coadministered drugs, or most morbidities (with the exception of renal failure and dialysis). TPMT phenotyping methods had coefficients of variation generally below 10 percent. TPMT genotyping reproducibility is generally between 95-100 percent. The sensitivity of genotyping to identify patients with low or intermediate TPMT enzymatic activity is imprecise, ranging from 70.70 to 82.10 percent (95 percent CI, lower bound range 37.90 to 54.00 percent; upper bound range 84.60 to 96.90 percent). Sensitivity of homozygous TPMT genotype to correctly identify patients with low to absent enzymatic activity was 87.10 percent (95 percent CI 44.30 to 98.30 percent). Genotyping specificity approached 100 percent. Leukopenia was significantly associated with low and intermediate enzymatic activity (low activity OR 80.00, 95 percent CI 11.5 to 559; and intermediate activity OR 2.96, 95 percent CI 1.18 to 7.42), and homozygous and heterozygous TPMT variant allele genotype (OR 18.60, 95 percent CI 4.12 to 83.60; and 4.62, 95 percent CI 2.34 to 9.16, respectively). In general, TPMT phenotyping costs less than genotyping, although estimates across studies are quite heterogeneous. CONCLUSIONS: There is insufficient direct evidence regarding the effectiveness of pretesting of TPMT status in patients with chronic autoimmune diseases. Indirect evidence confirms strong association of leukopenia with lower levels of TPMT activity and carrier genotype already established in the literature.

Degradation of circulating von Willebrand factor and its regulator ADAMTS13 implicates secreted Bacillus anthracis metalloproteases in anthrax consumptive coagulopathy.

Pathology data from the anthrax animal models show evidence of significant increases in vascular permeability coincident with hemostatic imbalances manifested by thrombocytopenia, transient leucopenia, and aggressive disseminated intravascular coagulation. In this study we hypothesized that anthrax infection modulates the activity of von Willebrand factor (VWF) and its endogenous regulator ADAMTS13, which play important roles in hemostasis and thrombosis, including interaction of endothelial cells with platelets. We previously demonstrated that purified anthrax neutral metalloproteases Npr599 and InhA are capable of cleaving a variety of host structural and regulatory proteins. Incubation of human plasma with these proteases at 37 degrees C in the presence of urea as a mild denaturant results in proteolysis of VWF. Also in these conditions, InhA directly cleaves plasma ADAMTS13 protein. Npr599 and InhA digest synthetic VWF substrate FRETS-VWF73. Amino acid sequencing of VWF fragments produced by InhA suggests that one of the cleavage sites of VWF is located at domain A2, the target domain of ADAMTS13. Proteolysis of VWF by InhA impairs its collagen binding activity (VWF:CBA) and ristocetin-induced platelet aggregation activity. In plasma from anthrax spore-challenged DBA/2 mice, VWF antigen levels increase up to 2-fold at day 3 post-infection with toxigenic Sterne 34F(2) strain, whereas VWF:CBA levels drop in a time-dependent manner, suggesting dysfunction of VWF instead of its quantitative deficiency. This conclusion is further supported by significant reduction in the amount of VWF circulating in blood in the ultra-large forms. In addition, Western blot analysis shows proteolytic depletion of ADAMTS13 from plasma of spore-challenged mice despite its increased expression in the liver. Our results suggest a new mechanism of anthrax coagulopathy affecting the levels and functional activities of both VWF and its natural regulator ADAMTS13. This mechanism may contribute to hemorrhage and thrombosis typical in anthrax.

Endotoxin fever in granulocytopenic rats: evidence that brain cyclooxygenase-2 is more important than circulating prostaglandin E(2).

PGE(2) is a recognized mediator of many fevers, and cyclooxygenase (COX) is the major therapeutic target for antipyretic therapy. The source, as well as the site of action of PGE(2), as an endogenous pyrogen, is widely accepted as being central, but PGE(2) in the circulation, possibly from leukocytes, may also contribute to the development of fever. However, bacterial infections are important causes of high fever in patients receiving myelosuppressive chemotherapy, and such fevers persist despite the use of COX inhibitors. In the study reported here, the febrile response to bacterial LPS was measured in rats made leukopenic by cyclophosphamide. A striking increase in LPS fever occurred in these granulocytopenic rats when compared with febrile responses in normal animals. Unlike LPS fever in normal rats, fever in granulocytopenic rats was neither accompanied by an increase in blood PGE(2) nor inhibited by ibuprofen. Both leukopenic and normal rats showed LPS-induced COX-2-immunoreactivity in cells associated with brain blood vessels. Furthermore, LPS induced an increase of PGE(2) in cerebrospinal fluid. Induction of COX-2-expression and PGE(2) production was inhibited by ibuprofen in normal but not in leukopenic rats. Although the results presented are, in part, confirmatory, they add new information to this field and open a number of important questions as yet unresolved. Overall, the present results indicate that, in contrast to immunocompetent rats, leukocytes and/or other mechanisms other than PGE(2) are implicated in the mechanisms restricting and reducing the enhanced febrile response to endotoxin in immunosuppressed hosts.

[The apoptosis marker enzyme poly-(ADP-ribose) polymerase (PARP) in systemic lupus erythematosus]. / Das Apoptose-Markerenzym Poly-(ADP-Ribose-)Polymerase (PARP) beim systemischen Lupus erythematodes.

The enzyme poly-(ADP-ribose) polymerase (PARP) is localized within the cell nucleus and catalyzes DNA-repair. During programmed cell death (apoptosis), PARP is enzymatically cleaved. Detection of the cleavage products is characteristic for apoptosis. In patients with systemic lupus erythematosus (SLE), the highly ordered signal transduction cascade of apoptosis is disturbed. SLE patients show reduced PARP activity . PARP cleavage products are mainly found in association with either antinuclear and/or anti-dsDNA antibodies. In addition, serum samples from SLE patients and other autoimmune diseases display anti-PAR and anti-PARP autoantibodies.

TPMT in the treatment of Crohn's disease with azathioprine.

Azathioprine induced profound myelosuppression linked to TPMT deficiency has now been documented in many patient groups, including those with Crohn's disease. At the start of azathioprine or mercaptopurine therapy, measurement of TPMT activity has a role in identifying the 1 in 300 patients who are at risk of severe myelosuppression when treated with standard thiopurine dosages. During the initial months of azathioprine therapy a knowledge of TPMT status warns of early bone marrow toxicity. In patients established on azathioprine these is no clear evidence to suggest that TPMT is predictive of clinical response or drug toxicity, indicating a role for TPMT in the prediction of early events rather than long term control. In patients with Crohn's disease on long term azathioprine therapy, it is clear that myelosuppression, particularly leucopenia, is caused by other factors in addition to variable TPMT activity and therefore monitoring of blood cell counts throughout treatment is essential.

Gabexate mesilate, a synthetic protease inhibitor, prevents compression-induced spinal cord injury by inhibiting activation of leukocytes in rats.

OBJECTIVE: Gabexate mesilate is a synthetic protease inhibitor capable of inhibiting both coagulation and cytokine production by monocytes. To investigate whether gabexate mesilate is useful for the prevention of posttraumatic spinal cord injury, we examined its effect on compression trauma-induced spinal cord injury in rats. DESIGN: Prospective, randomized, blinded, controlled study. SETTING: Research laboratory at a university medical center. SUBJECTS: Male Wistar rats weighing 300 to 350 g. INTERVENTIONS: Spinal cord injury was induced by applying a 20-g weight extradurally to the spinal cord at the level of the 12th thoracic vertebra for 20 mins. Spinal cord injury was evaluated by assessing the motor function of the rats 24 hrs posttrauma. The accumulation of leukocytes and histologic changes in the injured spinal cord tissue also were examined. Rats received gabexate mesilate (10 or 20 mg/kg i.p.) 30 mins before or after the compressive trauma. The effects of heparin or an inactive derivative of activated factor X (a selective inhibitor of thrombin generation) on compressive trauma-induced spinal cord injury also were examined. Leukocytopenia was induced by the administration of nitrogen mustard. MEASUREMENTS AND MAIN RESULTS: The motor disturbances observed following traumatic spinal cord compression, evaluated by Tarlov's score, and the accumulation of leukocytes in the injured tissue, evaluated by measuring tissue myeloperoxidase activity, were markedly reduced by leukocyte depletion induced by nitrogen mustard and by pre- or posttreatment of animals with gabexate mesilate. Neither heparin nor the inactive derivative of activated factor X prevented the motor disturbances and the accumulation of leukocytes. Histologic examination demonstrated that intramedullary hemorrhages observed 24 hrs after trauma at the 12th thoracic vertebra were significantly attenuated by nitrogen mustard-induced leukocytopenia and the administration of gabexate mesilate. CONCLUSIONS: The compression trauma-induced spinal cord injury demonstrated by this model was mainly mediated by leukocytes. Gabexate mesilate prevented spinal cord injury not by inhibiting coagulation, but by inhibiting the activation of leukocytes.