EV71:TLLcho virus murine model of enterovirus A71 neurological disease does not exhibit neurogenic pulmonary oedema.

Small animal models play an important role in investigating and revealing the molecular determinants and mechanisms underlying neuro-virulence of enterovirus A71 (EV-A71). In our previous study, we successfully developed two mouse cell-line replication competent EV-A71 strains (EV71:TLLm and EV71:TLLmv) which were capable of inducing neuro-invasion in BALB/c mice. The more virulent EV71:TLLmv exhibited ability to induce acute encephalomyelitis accompanied by neurogenic pulmonary oedema. EV71:TLLcho virus strain was generated from EV71:TLLm by a series of passages in CHO-K1 cells. EV71:TLLcho demonstrated a broader range of infectivity across various mammalian cell lines and exhibited complete cytopathic effects (CPE) within 48 hours post-inoculation in comparison to EV71:TLLm or EV71:TLLmv. EV71:TLLcho consistently yielded higher levels of viral replication at all time points examined. In comparison to EV71:TLLm, EV71:TLLcho consistently induced more severe disease and increased mortality in one-week old BALB/c mice. However, unlike mice challenged with EV71:TLLmv, none of the mice challenged with EV71:TLLcho progressed to severe acute encephalomyelitis and developed neurogenic pulmonary oedema.

Prognostic gene expression signature for high-grade serous ovarian cancer.

BACKGROUND: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. PATIENTS AND METHODS: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. RESULTS: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. CONCLUSION: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Evaluation of DNA methylation at imprinted DMRs in the spermatozoa of oligozoospermic men in association with MTHFR C677T genotype.

Altered DNA methylation has been previously identified in the spermatozoa of infertile men; however, the origins of these errors are poorly understood. DNA methylation is an epigenetic modification which is thought to play a fundamental role in male germline development. DNA methylation reactions rely on the cellular availability of methyl donors, which are primarily products of folate metabolism, where a key enzyme is methylenetetrahydrofolate reductase (MTHFR). The MTHFR C677T single nucleotide polymorphism (SNP) reduces enzyme activity and may potentially alter DNA methylation processes during germline development. The objective of this study was to determine whether altered DNA methylation in spermatozoa is associated with the MTHFR C677T SNP. DNA methylation was evaluated at the H19, IG-GTL2, and MEST imprinted differentially methylated regions in the spermatozoa of 53 men - 44 oligozoospermic men and nine fertile men with normal sperm parameters via bisulfite sequencing of sperm clones. The 44 infertile men were stratified by severity of oligozoospermia - three normal (>15 million spermatozoa/mL), eight moderate (5-15 million spermatozoa/mL), 23 severe (1-5 million spermatozoa/mL), and 10 very severe (<1 million spermatozoa/mL). MTHFR C677T SNP genotyping was conducted in a subset of 44 peripheral blood samples via restriction fragment length polymorphism. A total of three men - severe oligozoospermic and CT genotype - were found to be altered, which is defined as having ≥50% of their clones altered, where an altered clone was in turn defined as ≥50% of CpGs with incorrect DNA methylation patterns. The incidence of three altered men within the CT subgroup, however, was not significantly higher than the incidence in the CC subgroup. Taken together, altered DNA methylation in spermatozoa was not significantly associated with the MTHFR C677T SNP; however, there was a trend for higher incidence of alterations among severe oligozoospermic infertile men with CT genotypes.

Gene Microarray Analyses of Daboia russelli russelli Daboiatoxin Treatment of THP-1 Human Macrophages Infected with Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis and represents a potential bioterrorism threat. In this study, the transcriptomic responses of B. pseudomallei infection of a human macrophage cell model were investigated using whole-genome microarrays. Gene expression profiles were compared between infected THP-1 human monocytic leukemia cells with or without treatment with Daboia russelli russelli daboiatoxin (DRRDbTx) or ceftazidime (antibiotic control). Microarray analyses of infected and treated cells revealed differential upregulation of various inflammatory genes such as interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α), cyclooxygenase (COX-2), vascular endothelial growth factor (VEGF), chemokine C-X-C motif ligand 4 (CXCL4), transcription factor p65 (NF-kB); and several genes involved in immune and stress responses, cell cycle, and lipid metabolism. Moreover, following DRR-DbTx treatment of infected cells, there was enhanced expression of the tolllike receptor 2 (TLR-2) mediated signaling pathway involved in recognition and initiation of acute inflammatory responses. Importantly, we observed that highly inflammatory cytokine gene responses were similar in infected cells exposed to DRR-DbTx or ceftazidime after 24 h. Additionally, there were increased transcripts associated with cell death by caspase activation that can promote host tissue injury. In summary, the transcriptional responses during B. pseudomallei infection of macrophages highlight a broad range of innate immune mechanisms that are activated within 24 h post-infection. These data provide insights into the transcriptomic kinetics following DRR-DbTx treatment of human macrophages infected with B. pseudomallei.

The contribution of cardiovascular mortality to long term outcomes in a relatively young demographic following acute pulmonary embolism: a validation study.

BACKGROUND: Long-term studies following acute pulmonary embolism (PE) remain limited in the current era. A recent study from our collaborative group, in a contemporary adult population, showed substantially increased cardiovascular mortality following PE. We sought to evaluate the contribution of cardiovascular mortality to long-term outcomes in a different demographic that comprised of a significantly younger PE cohort. METHODS AND RESULTS: Demographic and clinical characteristics were retrospectively collected for this cohort, and similar methods and outcome measures were applied as detailed in the original study. We compared a population from a different metropolitan area (LH: Liverpool Hospital) to that from the original study (CRGH: Concord Hospital) over a similar time period. A total of 815 patients comprised this cohort with mean 5.3±3.8year follow-up. There were similar demographics between the two cohorts, though the mean age was significantly younger in LH group (60 vs 68years, p<0.001). Prior history of cardiovascular disease in the LH group was half of that present in the CRGH cohort. The overall mortality was 7.4% per patient-year. Patients with underlying cardiovascular disease when presenting with an acute PE had a 2.3-fold increased risk of death during follow-up compared to those without. Multivariate analysis showed that older age, male gender, malignancy, diabetes, cardiovascular disease and chronic pulmonary disease were independent predictors of post-discharge mortality. CONCLUSIONS: Despite our cohort being significantly younger with a lower incidence of pre-existing cardiovascular disease, cardiovascular disease was still a significant contributor to long-term outcomes and an important predictor of mortality following acute PE.

Xylan utilization regulon in Xanthomonas citri pv. citri Strain 306: gene expression and utilization of oligoxylosides.

Xanthomonas citri pv. citri strain 306 (Xcc306), a causative agent of citrus canker, produces endoxylanases that catalyze the depolymerization of cell wall-associated xylans. In the sequenced genomes of all plant-pathogenic xanthomonads, genes encoding xylanolytic enzymes are clustered in three adjacent operons. In Xcc306, these consecutive operons contain genes encoding the glycoside hydrolase family 10 (GH10) endoxylanases Xyn10A and Xyn10C, the agu67 gene, encoding a GH67 α-glucuronidase (Agu67), the xyn43E gene, encoding a putative GH43 α-l-arabinofuranosidase, and the xyn43F gene, encoding a putative ß-xylosidase. Recombinant Xyn10A and Xyn10C convert polymeric 4-O-methylglucuronoxylan (MeGXn) to oligoxylosides methylglucuronoxylotriose (MeGX3), xylotriose (X3), and xylobiose (X2). Xcc306 completely utilizes MeGXn predigested with Xyn10A or Xyn10C but shows little utilization of MeGXn. Xcc306 with a deletion in the gene encoding α-glucuronidase (Xcc306 Δagu67) will not utilize MeGX3 for growth, demonstrating the role of Agu67 in the complete utilization of GH10-digested MeGXn. Preferential growth on oligoxylosides compared to growth on polymeric MeGXn indicates that GH10 xylanases, either secreted by Xcc306 in planta or produced by the plant host, generate oligoxylosides that are processed by Xyn10 xylanases and Agu67 residing in the periplasm. Coordinate induction by oligoxylosides of xyn10, agu67, cirA, the tonB receptor, and other genes within these three operons indicates that they constitute a regulon that is responsive to the oligoxylosides generated by the action of Xcc306 GH10 xylanases on MeGXn. The combined expression of genes in this regulon may allow scavenging of oligoxylosides derived from cell wall deconstruction, thereby contributing to the tissue colonization and/or survival of Xcc306 and, ultimately, to plant disease.

Diagnostic investigation of parotid neoplasms: a 16-year experience of freehand fine needle aspiration cytology and ultrasound-guided core needle biopsy.

This study aimed to examine the diagnostic yield of fine needle aspiration cytology (FNAC) and ultrasound-guided core needle biopsy (USCB) in the diagnosis of parotid neoplasia. A 16-year retrospective analysis was performed of patients entered into our pathology database with a final diagnosis of parotid neoplasia. FNAC and USCB data were compared to surgical excision where available. One hundred and twenty FNAC, 313 USCB, and 259 surgical specimens were analyzed from 397 patients. Fifty-six percent of FNAC and 4% of USCB were non-diagnostic. One hundred and thirty-two (33%) patients had a final diagnosis made by USCB and did not undergo surgery. Surgery was performed in 257 (65%) patients, 226 (88%) of whom had a preoperative biopsy. Most lesions were benign, but there were 62 parotid and 13 haematological malignancies diagnosed; false-negative results were obtained in three FNAC and two USCB samples. The sensitivity and specificity of FNAC were 70% and 89%, respectively, and for USCB were 93% and 100%, respectively. This study represents the largest series of patients with a parotid neoplasm undergoing USCB for diagnosis. USCB is highly accurate with a low non-diagnostic rate and should be considered an integral part of parotid assessment.

Asymptomatic left ventricular dysfunction with long-term clozapine treatment for schizophrenia: a multicentre cross-sectional cohort study.

OBJECTIVES: Patients with schizophrenia treated with clozapine are at risk of acute myocarditis and dilated cardiomyopathy. However, there are no data on the prevalence of subclinical cardiomyopathy or its associations. METHODS: 100 consecutive patients with schizophrenia treated with clozapine for >1 year and without a history of cardiac pathology (group 1), 21 controls with a history of schizophrenia treated with non-clozapine antipsychotics for >1 year (group 2) and 20 controls without schizophrenia (group 3) were studied. Comprehensive evaluation by clinical examination, ECG, transthoracic echocardiography including left ventricular ejection fraction (LVEF) and global longitudinal strain (GLS) and biochemical profiles were performed. RESULTS: Patients with schizophrenia were of similar age, but had higher body mass index (BMI), rates of smoking and hyperlipidaemia than controls. Patients with schizophrenia had received clozapine or non-clozapine antipsychotics for a mean duration of 6.8±5.3 and 9.7±6.1 years, respectively. Patients taking clozapine demonstrated globally impaired LVEF (58.3%: group 1 vs 62.2%: group 2 vs 64.8%: group 3, p<0.001) and GLS (-16.7%: group 1 vs -18.6%: group 2 vs -20.2%: group 3, p<0.001). Moreover, LVEF was <50% in 9/100 (9%) patients receiving clozapine and in non-clozapine schizophrenia patients or healthy controls, but this was not statistically significantly different (analysis of covariance, p=0.19). Univariate analysis in patients taking clozapine found that impaired LV was not predicted by high-sensitivity troponin T, but was associated with features of the metabolic syndrome (including increased triglycerides, low high-density lipoprotein cholesterol (HDL-C), high-sensitivity C reactive protein and BMI), elevated neutrophil count, elevated heart rate, smoking and N-terminal probrain natriuretic peptide. In patients taking clozapine, multivariable analysis identified elevated neutrophil count and low HDL-C as the only independent predictors of impaired GLS. CONCLUSIONS: Asymptomatic mild LV impairment is common in patients with schizophrenia receiving long-term clozapine treatment and is associated with neutrophilia and low HDL-C.

Combination therapy with hepatocyte growth factor and oseltamivir confers enhanced protection against influenza viral pneumonia.

Frequent outbreaks caused by influenza viruses pose considerable public health threats worldwide. Virus-inflicted alveolar damage represents a major contributor of acute lung injury in influenza. We have previously demonstrated that hepatocyte growth factor (HGF) produced by macrophages enhances alveolar epithelial proliferation during influenza infection. Here, we investigated the therapeutic efficacy of recombinant human HGF (rhHGF) and an antiviral agent (oseltamivir) alone or in combination to treat influenza viral pneumonia in macrophage-depleted BALB/c mice. Combination therapy of infected mice significantly reduced lung pathology and mortality compared to other animal groups that received either treatment alone. Combination treatment with rhHGF induced alveolar type II (AT2) epithelial hyperplasia more prominently in the distal airways, evident by increased cells with double-positive staining for surfactant protein-C and proliferating cell nuclear antigen within the alveolar epithelial lining. Similarly, rhHGF supplementation also induced stem cell antigen-1 (SCA-1) transcriptional expression at 5 days post-infection (dpi), but mRNA levels of both SCA-1 and its receptor c-KIT were decreased by 10 dpi. Microarray and pathway analyses indicated that rhHGF administration may act by accelerating tissue repair and suppressing inflammatory processes to minimize damage by infection and to restore lung function by earlier repair. These results reveal that transient administration of rhHGF may confer synergistic effects in enhancing pulmonary repair by promoting AT2 cell proliferation. Thus, the combination of rhHGF and oseltamivir may represent a promising therapeutic option against influenza pneumonia to improve existing antiviral treatment regimens.

Evaluation of biopsy methods in the diagnosis of submandibular space pathology.

The aim of this study was to evaluate the performance of fine needle aspiration cytology (FNAC), ultrasound-guided core needle biopsy (USCNB), punch biopsy, and surgical excision biopsy in neoplasms presenting within the submandibular space. A retrospective analysis of all patients with a pathological diagnosis of a submandibular space neoplasm within a 12-year period (February 1999 to June 2011) was performed. Biopsy results were compared to histopathological diagnosis obtained from surgical excision biopsy. Eighty-one specimens from 44 patients met the search criteria (15 FNAC, 24 USCNB, 7 punch biopsy, and 35 surgical excision biopsy). The final diagnosis was established by USCNB, punch biopsy, or surgical excision biopsy and not by FNAC alone. Surgical excision biopsy was performed as a primary diagnostic (n = 8), secondary diagnostic (n = 15), or as a post-diagnostic therapeutic procedure (n = 12). Non-diagnostic results were: FNAC 11/15, USCNB 2/24, and punch biopsy 1/7. Diagnostic results were: FNAC 2/15, USCNB 20/24, and punch biopsy 5/7. No complications were reported. Although punch biopsy demonstrated good yield and accuracy, its use is restricted to a small cohort of patients. USCNB is a safe and accurate technique in the submandibular space, with a low non-diagnostic rate.

Acute pulmonary embolism during warfarin therapy and long-term risk of recurrent fatal pulmonary embolism.

The clinical characteristics and long-term outcomes of patients presenting with acute pulmonary embolism (PE) during treatment with warfarin have not been described. Clinical details of all patients admitted to a tertiary institution from 2000-2007 with acute PE were retrieved retrospectively, baseline warfarin status and the international normalised ratio (INR) were recorded, and their outcomes tracked using a statewide death registry. Of 923 patients with clearly documented warfarin status included in this study, 83 (9%) were taking warfarin. Mean (± standard deviation) day-1 INR of those taking warfarin was 2.3 ± 0.9, with 67% of patients therapeutically anti-coagulated (INR ≥2.0) at presentation (49 patients with INR <2.5 and 34 with INR ≥2.5). Patients taking warfarin on admission were more likely to have heart failure, atrial fibrillation and valvular heart disease, with similar prevalence of malignancy and ischaemic heart disease, compared to patients not on warfarin. Total mortality of the cohort (mean follow-up 4.0 ± 2.5 years) was 31.6% (in-hospital mortality 1.5%), and was similar between warfarin and no warfarin groups. There was however a greater than four-fold increased risk of post-discharge death due to recurrent PE for the patients taking warfarin on admission (hazard ratio [HR] 4.43, 95% confidence interval [CI] 1.36-14.42, p=0.01). Among patients taking warfarin on admission, day-1 INR <2.5 significantly increased long-term all-cause mortality compared to INR ≥2.5 (adjusted HR 2.51, 95% CI 1.08-5.86, p=0.03). In conclusion, patients presenting with PE during treatment with warfarin have an increased risk of death from recurrent PE. Admission INR appears to have independent long-term prognostic importance in these patients.

Acute coronary syndrome and stable coronary artery disease: are they so different? Long-term outcomes in a contemporary PCI cohort.

BACKGROUND: Patients undergoing percutaneous coronary intervention (PCI) for acute coronary syndromes (ACS) are known to have poorer short-term prognosis compared to stable coronary artery (CAD) patients undergoing elective PCI. Few studies have made direct comparison of long-term mortality between ACS and stable CAD patients undergoing PCI. The aim of our study was to compare the long-term mortality following PCI between patients with ACS and those with stable CAD. METHODS: We examined consecutive patients undergoing PCI with stenting at a tertiary referral hospital. Clinical, angiographic and biochemical data were collected and analysed. The primary outcome was all-cause mortality retrieved from the Statewide Death Registry database. RESULTS: Included were 1923 consecutive PCI patients (970 stable CAD and 953 ACS). The mean follow-up time was 4.1 years ± 1.8 years. In-hospital mortality was 1.4% overall, seen exclusively in patients with ACS (n=28, 2.9%). Post-discharge mortality was 6.7% among patients with stable CAD and 10.5% for ACS (P<0.01). Multivariate predictors of post-discharge deaths for both groups included age (HR 1.08 per year, P<0.001) and impaired renal function (HR 2.49, P<0.001). Following adjustment for these factors, an ACS indication for PCI was not associated with greater post-discharge mortality (adjusted HR 1.18: 0.85-1.64, P=0.32). CONCLUSIONS: Patients undergoing PCI following an ACS have higher long-term mortality to those with stable CAD, which is potentially explained by a greater prevalence of comorbidities. This suggests that for the ACS population, contemporary interventional and medical management strategies may effectively and specifically counter the adverse prognostic impact of coronary instability and myocardial damage.

Snake venom phospholipases A(2): a novel tool against bacterial diseases.

The majority of snake venom phospholipases A(2) (svPLA(2)s) are toxic and induce a wide spectrum of biological effects. They are cysteine-rich proteins that contain 119-134 amino acids and share similar structures and functions. About 50% of the residues are incorporated into α-helices, whereas only 10% are in ß-sheets. Fourteen conserved cysteines form a network of seven disulfide bridges that stabilize the tertiary structure. They show a high degree of sequence and structural similarity, and are believed to have a common calcium- dependent catalytic mechanism. Additionally, svPLA(2)s display an array of biological actions that are either dependent or independent of catalysis. The PLA(2)s of mammalian origin also exert potent bactericidal activity by binding to anionic surfaces and enzymatic degradation of phospholipids in the target membranes, preferentially of Gram-positive species. The bactericidal activity against Gram-negatives by svPLA(2) requires a synergistic action with bactericidal/permeability-increasing protein (BPI), but is equally dependent on enzymatic- based membrane degradation. Several hypotheses account for the bactericidal properties of svPLA(2)s, which include "fatal depolarization" of the bacterial membrane, creation of physical holes in the membrane, scrambling of normal distribution of lipids between the bilayer leaflets, and damage of critical intracellular targets after internalization of the peptide. The present review discusses several svPLA(2)s and derived peptides that exhibit strong bactericidal activity. The reports demonstrate that svPLA(2)-derived peptides have the potential to counteract microbial infections. In fact, the C-terminal cationic/hydrophobic segment (residues 115-129) of svPLA(2)s is bactericidal. Thus identification of the bactericidal sites in svPLA(2)s has potential for developing novel antimicrobials.

Comparative seroepidemiology of pertussis, diphtheria and poliovirus antibodies in Singapore: waning pertussis immunity in a highly immunized population and the need for adolescent booster doses.

BACKGROUND: We assessed the seroepidemiology of pertussis, diphtheria and poliovirus antibodies in a cohort of highly immunized children, together with the burden of these diseases in Singapore. METHODS: Hospital residual sera collected between August 2008 and July 2010 from 1200 children aged 1-17 years were tested for the prevalence of IgG antibodies against Bordetella pertussis, diphtheria toxoid, and all three poliovirus types by enzyme-linked immunosorbent assays. RESULTS: We found an overall seroprevalence of 99.4% (95% CI 98.8-99.7%) for diphtheria, and 92.3% (95% CI 90.6-93.6%) for poliomyelitis, along with no indigenous cases of these diseases since 1993. However, the seroprevalence for pertussis was 60.8% (95% CI 58.0-63.5%) only. Among the subjects who had completed three doses of pertussis vaccination by the age of 2 years (n=1092), the pertussis seroprevalence was 85.0% (95% CI 79.7-89.2%) in those who received the last vaccination within a year before the study, and it decreased to 75.0% (95% CI 64.5-83.2%) and 63.1% (95% CI 50.9-73.8%) in those who had the last vaccination 1 year and 2 years before the study, respectively. The seroprevalence remained at about 50% for those whose last pertussis vaccination was administered 4 years and longer before the study. CONCLUSIONS: The high seroprevalence for poliomyelitis and diphtheria confer solid herd immunity to eliminate these diseases in Singapore. In contrast, immunity against pertussis waned considerably over time, and routine boosters should be given to adolescents to ensure sustained immunity against pertussis.

Antimicrobial proteins from snake venoms: direct bacterial damage and activation of innate immunity against Staphylococcus aureus skin infection.

The innate immune system is the first line of defense against microbial diseases. Antimicrobial proteins produced by snake venoms have recently attracted significant attention due to their relevance to bacterial infection and potential development into new therapeutic agents. Staphylococcus aureus is one of the major human pathogens causing a variety of infections involving pneumonia, toxic shock syndrome, and skin lesions. With the recent emergence of methicillin (MRSA) and vancomycin (VRSA) resistance, S. aureus infection is a serious clinical problem that will have a grave socio-economic impact in the near future. Although S. aureus susceptibility to innate antimicrobial peptides has been reported recently, the protective effect of snake venom phospholipase A2 (svPLA2) proteins on the skin from S. aureus infection has been understudied. This review details the protective function of svPLA2s derived from venoms against skin infections caused by S. aureus. We have demonstrated in vivo that local application of svPLA2 provides complete clearance of S. aureus within 2 weeks after treatment compared to fusidic acid ointment (FAO). In vitro experiments also demonstrate that svPLA2 proteins have inhibitory (bacteriostatic) and killing (bactericidal) effects on S. aureus in a dose-dependant manner. The mechanism of bacterial membrane damage and perturbation was clearly evidenced by electron microscopic studies. In summary, svPLA2s from Viperidae and Elapidae snakes are novel molecules that can activate important mechanisms of innate immunity in animals to endow them with protection against skin infection caused by S. aureus.

Phase 1 dose-escalation study of CP-690 550 in stable renal allograft recipients: preliminary findings of safety, tolerability, effects on lymphocyte subsets and pharmacokinetics.

CP-690 550 inhibits Janus kinase 3 with nanomolar potency. In this dose-escalation study, we assessed the safety, tolerability, effects on lymphocyte subsets, and pharmacokinetics of CP-690 550 when coadministered with mycophenolate mofetil in stable renal allograft recipients for 28 days. Twenty-eight patients were enrolled. Six patients received CP-690 550 5 mg twice daily (BID), 6 patients received 15 mg BID, 10 patients received 30 mg BID, and 6 patients received placebo. The most frequent adverse events were infections and gastrointestinal (abdominal pain, diarrhea, dyspepsia, and vomiting). CP-690 550 15 mg BID and 30 mg BID were associated with a mean decrease in hemoglobin from baseline of 11% and a mean decrease in absolute natural killer cell counts of 50%. CP-690 550 30 mg BID was also associated with a mean increase in absolute CD19(+) B-lymphocytes of 130%. There were no changes in the number of neutrophils, total lymphocytes, platelets, or CD4(+) or CD8(+) T cells; clinical chemistry; vital signs; or electrocardiograms from the pretreatment baseline. Administration of CP-690 550 without a concomitant calcineurin inhibitor resulted in CP-690 550 exposures consistent with previous studies in nontransplant subjects. Additional dose-ranging studies are warranted to evaluate the safety and efficacy of CP-690 550 in renal transplant recipients over longer treatment duration.