Changes in fecal microbiota with CFTR modulator therapy: A pilot study.

Studies have demonstrated that people with CF with pancreatic insufficiency (PI) have fecal dysbioses. Evidence suggests the causes of these dysbioses are multifactorial, and that important drivers include antibiotic exposure, dietary intake, and CF gastrointestinal tract dysfunction, including nutrient malabsorption. In this pilot study, we tested whether initiation of the CFTR modulator treatments ivacaftor (in a cohort of pancreatic sufficient (PS) people with CF and an R117H CFTR variant) or lumacaftor/ivacaftor (in a cohort of PI people with CF and an F508del variant) changed fecal measures of malabsorption or fecal microbiomes. While we identified no statistically significant fecal changes with either treatment, we detected trends in the PI cohort when initiating lumacaftor/ivacaftor towards decreased fecal fat content and towards fecal microbiomes that more closely resembled the fecal microbiota of people without PI. While these findings support a model in which nutrient malabsorption resulting from CF-induced PI drives fecal dysbiosis, they must be validated in future, larger studies of fecal microbiome and malabsorption outcomes with highly effective CFTR modulator therapies.

AZA-MS: a novel multiparameter mass spectrometry method to determine the intracellular dynamics of azacitidine therapy in vivo.

The cytidine analogue, 5-azacytidine (AZA; 5-AZA-cR), is the primary treatment for myelodysplastic syndrome and chronic myelomonocytic leukaemia. However, only ~50% of treated patients will respond to AZA and the drivers of AZA resistance in vivo are poorly understood. To better understand the intracellular dynamics of AZA upon therapy and decipher the molecular basis for AZA resistance, we have developed a novel, multiparameter, quantitative mass spectrometry method (AZA-MS). Using AZA-MS, we have accurately quantified the abundance of the ribonucleoside (5-AZA-cR) and deoxyribonucleoside (5-AZA-CdR) forms of AZA in RNA, DNA and the cytoplasm within the same sample using nanogram quantities of input material. We report that although AZA induces DNA demethylation in a dose-dependent manner, it has no corresponding effect on RNA methylation. By applying AZA-MS to primary bone marrow samples from patients undergoing AZA therapy, we have identified that responders accumulate more 5-AZA-CdR in their DNA compared with nonresponders. AZA resistance was not a result of impaired AZA metabolism or intracellular accumulation. Furthermore, AZA-MS has helped to uncover different modes of AZA resistance. Whereas some nonresponders fail to incorporate sufficient 5-AZA-CdR into DNA, others incorporate 5-AZA-CdR and effect DNA demethylation like AZA responders, but show no clinical benefit.

JC polyomavirus viremia and progressive multifocal leukoencephalopathy in human leukocyte antigen-sensitized kidney transplant recipients desensitized with intravenous immunoglobulin and rituximab.

BACKGROUND: Desensitization (DES) with intravenous immunoglobulin (IVIG) + rituximab is effective, safe, and increases the transplantation rate in human leukocyte antigen-sensitized patients. However, reports of progressive multifocal leukoencephalopathy (PML) caused by JC polyomavirus (JCPyV) in autoimmune patients treated with rituximab is concerning. Here, we report on the JCPyV viremia and PML status in kidney transplant patients with/without DES (non-DES). METHODS: In total 1195 and 699 DNA samples from plasma in 117 DES (78% lymphocyte-depleting [LyD] induction) and 100 non-DES patients (45% LyD), respectively, were submitted for JCPyV-polymerase chain reaction. Results were compared in both groups. RESULTS: No patients in either DES or non-DES developed PML or presented with any neurological symptoms. The JCPyV viremia rate was similar in DES and non-DES patients (3/117 vs. 9/100, P = 0.07). The JCPyV levels were low (median peak levels, 1025 copies/mL) and JCPyV viremia was observed only once during the study period in most patients. All 3 DES patients with JCPyV(+) received 1 dose rituximab and no DES patients with >1 dose rituximab showed JCPyV(+). All 3 JCPyV(+) DES patients received LyD induction, while only 2 of 9 JCPyV(+) non-DES patients did so, and the remaining 7 received non-LyD or no induction. JCPyV in leukocyte was mostly negative in DES and non-DES patients. Immunosuppression in patients with or without JCPyV(+) was similar. BK polyomavirus viremia was observed more commonly in patients with JCPyV(+) than in those without (P < 0.02). CONCLUSIONS: Patients with IVIG + rituximab DES followed by transplantation with LyD induction and additional rituximab rarely show JCPyV viremia and appear at low risk for PML.

A multicenter phase 1 study of PX-866 in combination with docetaxel in patients with advanced solid tumours.

BACKGROUND: This phase I, dose-finding study determined the safety, maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), pharmacokinetics, and antitumour activity of PX-866, a phosphatidylinositol 3-kinase inhibitor, combined with docetaxel in patients with incurable solid tumours. METHODS: PX-866 was administered at escalating doses (4-8 mg daily) with docetaxel 75 mg m⁻² intravenously every 21 days. Archived tumour tissue was assessed for potential predictive biomarkers. RESULTS: Forty-three patients were enrolled. Most adverse events (AEs) were grade 1 or 2. The most frequent study drug-related AE was diarrhoea (76.7%), with gastrointestinal disorders occurring in 79.1% (docetaxel-related) and 83.7% (PX-866-related). No dose-limiting toxicities were observed. The RP2D was 8 mg, the same as the single-agent MTD. Co-administration of PX-866 and docetaxel did not affect either drug's PKs. Best responses in 35 evaluable patients were: 2 partial responses (6%), 22 stable disease (63%), and 11 disease progression (31%). Eleven patients remained on study for >180 days, including 8 who maintained disease control on single-agent PX-866. Overall median progression-free survival (PFS) was 73.5 days (range: 1-569). A non-significant association between longer PFS for PIK3CA-MUT/KRAS-WT vs PIK3CA-WT/KRAS-WT was observed. CONCLUSION: Treatment with PX-866 and docetaxel was well tolerated, without evidence of overlapping/cumulative toxicity. Further investigation with this combination is justified.

Analysis of subcutaneous (SQ) alemtuzumab induction therapy in highly sensitized patients desensitized with IVIG and rituximab.

Here we report on our experience with subcutaneous (SQ) Alemtuzumab in an uncontrolled study in highly HLA-sensitized patients (HS). From 3/05-4/07, 54 HS patients received Alemtuzumab 30 mg SQ as induction. Patient and graft survival, AR episodes, serum creatinines, absolute lymphocyte counts, monthly PCR monitoring for viruses, AE/SAEs and infectious complications were monitored. No patient to date has developed acute injection-related reactions after SQ Alemtuzumab; however, bone marrow suppression was occasionally seen requiring reduction or elimination of mycophenolate mofetil approximately 1-2 months posttransplant. Patient and graft survival at 12 M was 98%/96%, respectively. AR episodes occurred in 35% with 20% being C4d+ AMR. Mean SCrs at 12 M were 1.4 +/- 0.3 mg/dL. The nadir ALC was 0.17 +/- 0.19 within 24 h and sustained up to 365 days posttransplant. Infections occurred in eight patients (five with polyoma BK viremia [PBK], one CMV/PBK and two CMV viremia). SQ Alemtuzumab was well tolerated and resulted in prolonged lymphocyte depletion. Compared to our previous experience with daclizumab and rabbit ATG induction in HS patients, single-dose SQ Alemtuzumab was more cost effective, showed similar infection rates and did not reduce the AMR rates posttransplant. Although uncontrolled, these observations suggest that induction therapy with Alemtuzumab appears feasible and indeed promising, but awaits more definitive study.

Effect of induction therapy protocols on transplant outcomes in crossmatch positive renal allograft recipients desensitized with IVIG.

Here we retrospectively examine the efficacy of two antibody induction regimens using Zenapax or Thymoglobulin in patients with positive complement-dependent cytotoxicity crossmatches (CDC-CMXs) desensitized with IVIG (intravenous immunoglobulin). Between January 1999 and March 2005, 97 patients with (+) CDC-CMXs received kidney transplants (43 deceased donors/54 living donors). All patients received at least 2 g/kg IVIG (maximum four doses) until an acceptable CMX was obtained. Patients were divided into two groups: 1. IVIG + Zenapax (n = 58), 2. IVIG + Thymoglobulin (n = 39). A total of 94% of patients in Group 1 and 84% in G2 have at least 2 years of follow up. Patient and graft survival was 96%/84% in Group 1 and 100%/90% in Group 2, p = NS. The number and severity of AR episodes were similar (36% Group 1 vs. 31% Group 2, p = NS) as was the incidence of C4d (+) antibody-mediated rejection (AMR) (Banff Grade II/III) (22% Group 1 vs. 21% Group 2). Mean serum creatinines (SCrs) at 24 months were similar (Group 1: 1.4 +/- 0.7 vs. G2: 1.5 +/- 0.7 mg/dL). Induction therapy with Zenapax or Thymoglobulin results in excellent patient, graft survival and graft function at 2 years. There was no increased risk of viral infections or malignancies with either agent. Neither agent was effective in reducing the incidence of AMR.

Effects of angiotensin II on the acrosome reaction in equine spermatozoa.

Angiotensin II is a hormone with a wide array of physiological effects that exerts its effect via interaction with two major subtypes of receptor. The results of this study show that angiotensin II (from 1 to 100 nmol l(-1)) initiates acrosomal exocytosis in equine spermatozoa that have undergone capacitation in vitro in a TALP-TEST (Tyrode's albumin lactate pyruvate; 188.7 mmol TES l(-1), 84.8 mmol Tris l(-1)) buffer with cAMP. The acrosome reaction and sperm viability were assessed with fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) and Hoechst 33258, respectively. The initiation of the acrosome reaction by angiotensin II was strongly inhibited by losartan, a specific angiotensin II type 1 receptor antagonist. Although angiotensin II as well as progesterone both initiated the acrosome reaction in equine spermatozoa, there was no synergistic effect when both agonists were added simultaneously. Initiation of acrosomal exocytosis by angiotensin II was accompanied by a rapid and transient calcium influx that was assessed in capacitated spermatozoa loaded with Fura-2AM. In addition, the angiotensin II-mediated calcium influx was inhibited when spermatozoa were preincubated with losartan. Western blotting with an antibody against angiotensin II type 1 receptor detected a major sperm protein of 60 kDa. Indirect immunofluorescence of non-capacitated spermatozoa with the angiotensin II type 1 receptor antibody revealed labelling in the midpiece and tail. In capacitated spermatozoa, the angiotensin II type 1 receptor was localized mainly over the anterior region of the sperm head, the equatorial segment and occasionally on the postacrosomal region in addition to the sperm tail. In conclusion, this study demonstrated the ability of angiotensin II to stimulate the acrosome reaction in capacitated equine spermatozoa. This effect is mediated via the angiotensin II type 1 receptor and is accompanied by an increase in intracellular calcium.

Coronary calcium and atherosclerosis by ultrafast computed tomography in asymptomatic men and women: relation to age and risk factors.

We evaluated 675 men and 190 women who had no symptoms or history of clinical CHD, to determine the prevalence and risk factor correlates of CAC deposits as a marker of atherosclerosis. Measurements were taken noninvasively by ultrafast CT. The presence and extent of CAC deposits as measured by ultrafast CT was determined in all subjects, who also received personal and family medical history and risk factor questionnaire. The prevalence of CAC deposits increased significantly with age, ranging from 15% and 30% in men and women, respectively, < 40 years of age to 93% and 75% in those aged > or = 70 years. Prevalence and total score also increased by the number of risk factors present, although in those aged > 60 years a high prevalence (> 80% in men) of calcium was present regardless of the presence of risk factors. In multiple logistic regression, age, male gender, hypertension, diabetes, hypercholesterolemia, and obesity were independently associated with CAC deposits. These results suggest a high prevalence of atherosclerosis with increasing age and the presence of risk factors in men and women who have no symptoms. Studies to determine the prognostic value of CAC in individuals with no symptoms are needed to determine which populations may benefit most from CAC deposit screening.

Detection of coronary artery calcium by ultrafast computed tomography and its relation to clinical evidence of coronary artery disease.

Ultrafast computed tomography (CT) was used to evaluate the relation of coronary artery calcium, a marker of atherosclerosis, with a reported history of coronary artery disease (CAD) in 928 men and 290 women (mean age 53 +/- 10 years; 11% with previous CAD). Total calcium score was calculated as the sum of each lesion-specific score, calculated as the product of pixel area and density > 130 Hounsfeld units. Total score was 3 to 6 times greater (p < 0.01) and the probability of coronary artery calcium 30 to 40% greater (p < 0.01) in patients with a reported history of myocardial infarction, positive angiography, bypass surgery or angioplasty. From score cutoffs ranging from 1 to 500 for defining calcium, a negative test was accurate 93 to 98% of the time in ruling out CAD, whereas specificity increased from 43 to 93%; however, sensitivity decreased from 92 to 42%. A score cutoff of 50 showed modest sensitivity (78%) and specificity (71%); however, the predictive value for CAD from a positive test remained low (< or = 40%), regardless of score cutoff. From multiple logistic regression, total score was also an independent indicator of CAD after considering any effects due to age, sex and other CAD risk factors. Further study is needed to document the long-term prognostic use of coronary calcium screening, including criteria that best project future risk of CAD.

Prevalence and sociobehavioral correlates of tobacco use among Hispanic children: the Tobacco Resistance Activity Program.

To examine tobacco use patterns and its sociobehavioral correlates among Hispanic in-school youth, a tobacco use and knowledge survey was administered to 660 children from fourth through sixth grade classes, of which 69% (n = 453) were of Hispanic origin. Male Hispanics were more exposed to previous tobacco use (38% vs. 20%, p < .01), reported more current smoking, had more smoking friends, received more cigarette offers, and wanted to try a cigarette compared to females (all p < .05). Also, more previous alcohol use was noted in the previous tobacco users, indicating its potential as a "gateway drug." From multivariate analyses, being offered cigarettes was the strongest environmental indicator of previous tobacco usage, followed by having adult smokers in the house, smoking friends, (all p < .01), and being around other smoking youth (p < .05). This study offers additional knowledge about factors that may cause Hispanic youth to initiate smoking, and suggests the need to address peer and social influences in school-based substance abuse programs.