Experience of islet isolation without neutral protease supplementation

BACKGROUND: We have reported improved islet isolation outcomes using a new digestion protocol where the pancreas is perfused only with collagenase, and neutral protease (NP) is administered during the digestion phase. Since the inception of this protocol, we have had some cases where administration of NP was not required. Our new protocol was utilized in 94 islet isolations. The timing of adding NP was dependent on the progression of digestion but in 10 cases the progression was rapid and most islets in the assessment samples were free from the exocrine tissue. As a result NP was not added at all for these isolations (no-NP group). In the remaining 84 isolations, NP was added during digestion phase (control group). RESULTS: Pancreata in the each group were digested with a similar collagenase dose. Digestion time was shorter in the no-NP (15.0±1.8 vs 19.5±0.6 min, P=0.004). At post-digestion, the no-NP had fewer trapped islets (10.9±2.8 vs 28.1±2.4%, P=0.009). Post-purification islet yield was similar (355±45 x10 ( 3) vs 318±17 x10 ( 3) IE, P=0.29). Five preparations in the no-NP were used for clinical transplantation, leading to a 64.3±15.2% reduction in insulin usage. Interestingly, cold ischemia time was longer in the no-NP (10.3±0.9 vs 7.9±0.4 h, P=0.04). One particular collagenase lot having the highest NP activity contamination was used in 7 isolations in the no-NP. Our experience indicates that supplementation of collagenase with NP is not always necessary for effective isolation. Cold ischemia time and NP contamination should be evaluated for optimal NP dosage.     

Molecular genetic rearrangements distinguish pre- and post-bone marrow transplantation lymphoproliferative processes

Chronic myelocytic leukemia (CML) may display a lymphoproliferative phase (lymphoid blast crisis) that is generally of B cell phenotype. Since lymphoproliferative disorders may occur following bone marrow transplantation (BMT), it may be difficult to distinguish posttransplant relapse of CML lymphoid blast crisis from de novo lymphoproliferation. Lymphoid blast crisis cells from a patient with CML displayed immunoglobulin heavy chain gene (C mu) rearrangement before BMT. Following BMT the patient developed a lymphoproliferative disorder involving multiple organs. Clonal rearrangement of C mu was demonstrated in several involved tissues. The rearranged C mu restriction fragment was distinct from that displayed before BMT. Additionally, rearrangement of the breakpoint cluster region (bcr) was demonstrated in the pretransplant blast crisis sample, but not in the posttransplant lymphoproliferation samples, thus confirming that these lymphoproliferative disorders were distinct. Molecular genetic techniques offer powerful diagnostic tools for monitoring the course of patients with CML undergoing BMT.     

Effects of human FLT3 ligand on myeloid leukemia cell growth: heterogeneity in response and synergy with other hematopoietic growth factors

A novel hematopoietic growth factor for primitive hematopoietic progenitor cells, the ligand for the flt3/flk2 receptor, (FL), has been recently purified and its gene has been cloned. In the present study, we investigated the effects of FL on the proliferation and differentiation of normal and leukemic myeloid progenitor cells. We demonstrate that FL is a potent stimulator of the in vitro growth of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 3 (IL-3), or G-CSF-dependent granulocyte-macrophage committed precursors from Lin- CD34+ bone marrow cells of normal donors. By contrast, FL does not affect the growth of erythroid-committed progenitors even in the presence of erythropoietin. The effect of FL on the proliferation and on the in vitro growth of clonogenic leukemic precursor cells was studied in 54 acute myeloid leukemia (AML) cases. Fresh leukemia blasts from 36 of 45 patients with AML significantly responded to FL without any relation to the French-American-British (FAB) subtype. FL stimulated the proliferation of leukemic blasts in a dose-dependent fashion. Synergistic activities were seen when FL was combined with G-CSF, GM-CSF, IL-3, or stem cell factor (SCF). FL as a single factor induced or increased significantly colony formation by clonogenic precursor cells from 21 of 24 patients with AML. In the presence of suboptimal and optimal concentrations of G-CSF, GM-CSF, IL3, SCF, or a combination of all factors, FL strongly enhanced the number of leukemic colonies (up to 18-fold). We also evaluated the induction of tyrosine phosphorylated protein on FL stimulation in fresh AML cells. We demonstrate that, on FL stimulation, a band of phosphorylated protein(s) of about 90 kD can be detected in FL- responsive, but not in FL-unresponsive cases. This study suggests that FL may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor with other cytokines.     

Clinical diagnosis of sleep apnea based on single night of polysomnography vs. two nights of polysomnography

Purpose  The purpose of this study was to investigate apnea–hypopnea index (AHI) across two polysomnographies (PSGs) to examine AHI variability and impact on clinical diagnosis. Materials and methods  Two-night PSGs of 193 sleep clinic patients were reviewed, and the AHI variability was analyzed. Anonymized records from five patients with significant night-to-night AHI variability were used in this study: the two-night PSGs from two patients were represented as four individual PSGs; the two-night PSG for two others were represented as being obtained from two different sleep clinics; the last patient’s PSG was shown as a two-night study. Twenty-two sleep experts attending the Associated Professional Sleep Societies meeting were recruited to make diagnoses based on the PSGs. They were told that the PSGs were from seven patients: four with single-night PSG; two with two PSGs, each one from a different clinic; and one patient with a two-night PSG. Results  Twenty-one percent of the 193 sleep clinic patients had a nightly PSG AHI variability of greater than 5. Forty-eight percent of all patients had a significantly higher AHI on the first night, and 41% had a significantly higher AHI on the second night. Using an AHI > 15 diagnostic criteria, sleep apnea would have been undetected in 20% (n = 39) of patients due to low AHI on one night. Furthermore, 13% of all patients had a more severe sleep apnea classification based on the second night of PSG. For the seven cases, 27–36% of sleep experts failed to identify sleep apnea especially when presented with the PSG containing the lower AHI. Incidences of missed sleep apnea diagnoses were reduced to 15–18% when information from two PSGs was presented to the sleep experts. Conclusions  Utilizing a large patient population, this study supports the significant night-to-night variability in PSG respiratory variables. Identification of sleep apnea in some patients is reduced when sleep experts are provided with only one PSG recording. The clinical implication is that about 13% of sleep clinic patients might benefit from a second night of PSG. Disclosure statement: This study did not receive external funding.     

Molecular and cytotoxic effects of camptothecin, a topoisomerase I inhibitor, on trypanosomes and Leishmania.

Parasites pose a threat to the health and lives of many millions of human beings. Among the pathogenic protozoa, Trypanosoma brucei, Trypanosoma cruzi, and Leishmania donovani are hemoflagellates that cause particularly serious diseases (sleeping sickness, Chagas disease, and leishmaniasis, respectively). The drugs currently available to treat these infections are limited by marginal efficacy, severe toxicity, and spreading drug resistance. Camptothecin is an established antitumor drug and a well-characterized inhibitor of eukaryotic DNA topoisomerase I. When trypanosomes or leishmania are treated with camptothecin and then lysed with SDS, both nuclear and mitochondrial DNA are cleaved and covalently linked to protein. This is consistent with the existence of drug-sensitive topoisomerase I activity in both compartments. Camptothecin also inhibits the incorporation of [3H]thymidine in these parasites. These molecular effects are cytotoxic to cells in vitro, with EC50 values for T. brucei, T. cruzi, and L. donovani, of 1.5, 1.6, and 3.2 microM, respectively. For these parasites, camptothecin is an important lead for much-needed new chemotherapy, as well as a valuable tool for studying topoisomerase I activity.     

Retrospective comparison of filgrastim plus plerixafor to other regimens for remobilization after primary mobilization failure: clinical and economic outcomes

We performed a retrospective analysis to evaluate clinical and economic outcomes in patients receiving remobilization therapy after primary mobilization failure. Our primary endpoint was to compare filgrastim plus plerixafor to other regimens in their ability to collect a target cell dose of at least 2 million CD34+ cells/kg (cumulative). Of 96 consecutive patients who failed their primary mobilization therapy and in whom a second mobilization was attempted, remobilization consisted of filgrastim plus plerixafor (n = 38), filgrastim with or without sargramostim (n = 43), or chemotherapy plus filgrastim (n = 15), 84% of filgrastim/plerixafor patients were able to collect at least 2 million CD34+ cells/kg from both mobilizations, compared to 60% of patients mobilized with chemotherapy/filgrastim and 79% of the filgrastim ± sargramostim patients (P = 0.17). However, when combined with cells collected from the first mobilization, 53% of filgrastim/plerixafor patients reached the target of 2 million CD34+ cells in one apheresis, compared to 20% of those receiving chemotherapy/filgrastim and 28% of those receiving filgrastim ± sargramostim (P = 0.02). Resource utilization, mobilization drug costs, clinical care costs, and total costs were significantly different. We conclude that while filgrastim/plerixafor is the most efficient remobilization strategy, those clinical benefits may not translate into lower cost, especially when multiple days of plerixafor administration are required.     

Imbalance of Apoptosis and Cell Proliferation Contributes to the Development and Persistence of Emphysema

Background  

We postulate that in adults there is an established lung structure maintenance program and that lung alveolar septal cells are undergoing both continuous apoptosis and proliferation. Whereas lung cell apoptosis has been recognized in human emphysema, little is known about cell proliferation.     

Superiority of mitral valve repair in surgery for degenerative mitral regurgitation

OBJECTIVES: We aimed to assess the influence of type of operation on outcomein degenerative mitral regurgitation. METHODS: We compared outcomes in 278 consecutive patients who underwentmitral valve repair (167 patients), replacement with subvalvularpreservation (22 patients) and without subvalvular preservation(89 patients) for degenerative mitral regurgitation. RESULTS: There was a trend towards lower mortality with repair and replacementwith subvalvular preservation compared to replacement withoutsubvalvular preservation. Thirty-day mortality was 1·2%vs 0·0% vs 4·7% (ns) respectively. Six-year survivalwas, respectively, 67·8±7·4% (P=0·088)vs 80·8±11·0% (P=0·25 vs 63·3±5·9%for all-cause death, 78·5±6·8% (P=0·063)vs 95·5±4·4% (P=0·092) vs 67·6±5·9%for all complication-related death and 80·5±6·9%(P=0·076) vs 100·0±0·0% (P=0·045)vs 72· ± 5·8% for complication-relateddeath due to myocardial failure. Multivariate analysis confirmedindependent beneficial effects from repair compared to replacementwithout subvalvular preservation on complication-related death(hazard ratio 0·42, P=0·010) and death from myocardialfailure (hazard ratio 0·40 P=0·014), and fromrepair compared to mechanical replacement on thromboembolism(hazard ratio 0·45, P=0·029) and anticoagulation-relatedhaemorrhage (hazard ratio 0·19, P=0·026). CONCLUSIONS: Mitral valve repair is superior to replacement. The greatestsurvival advantage is in reduced mortality from myocardial failure.Repair should be the operation of choice for degenerative mitralregurgitation.     

Transthoracic Doppler echocardiographic analysis of phasic coronary blood flow velocity in hypertrophic cardiomyopathy.

OBJECTIVE: To use transthoracic Doppler echocardiography to assess coronary blood flow non-invasively in patients with hypertrophic cardiomyopathy. DESIGN: High frequency transthoracic Doppler echocardiography was used to assess resting phasic coronary velocity patterns in patients with hypertrophic cardiomyopathy and to define the relation between coronary flow patterns and clinical, echocardiographic, and haemodynamic manifestations of this condition. SETTING: A tertiary referral cardiothoracic centre. METHODS: Fifteen patients (10 men and five women, mean (SD) age 49 (10.3) years) with asymmetric hypertrophic cardiomyopathy underwent high frequency (5 MHz) transthoracic Doppler echocardiographic assessment of the left anterior descending coronary artery. In addition, standard two dimensional echocardiography was performed. The results were compared with 16 normal participants (nine men and seven women, mean age 61.2 (10.7) years) who had no evidence of cardiac disease. RESULTS: Biphasic diastolic predominant coronary artery blood velocity profiles were obtained in all patients and controls. Systolic peak blood velocity and velocity time integral were significantly reduced in the hypertrophic cardiomyopathy group compared with controls (11.3 (15.8) cm/s and 1.09 (1.78) cm v 20.5 (13.1) cm/s and 4.23 (2.80) cm, respectively, P < 0.05). A reversed pattern of systolic blood flow velocity was found in three patients with severe anterior wall and septal hypertrophy. During diastole there was prolongation of the diastolic acceleration (203 (53) ms v 110 (60) ms in controls, P < 0.05) and deceleration times (487 (200) ms v 210 (90) ms in controls, P < 0.05). There was no significant difference between those with and without symptoms or a left ventricular outflow tract gradient. CONCLUSIONS: Patients with hypertrophic cardiomyopathy have abnormal systolic and diastolic coronary flow profiles at rest when measured by transthoracic echocardiography.     

Gender differences in the responsiveness of the sex-dependent isoforms of hepatic P450 to the feminine plasma growth hormone profile

Most of the constitutive hepatic P450 isoforms expressed in the rat exhibit dramatic gender differences. Whereas only male hepatocytes contain CYP2A2, 2C11, and 3A2, only female hepatocytes express CYP2C12 and 3- to 4-fold greater levels of CYP2C7. This sexually dimorphic expression of hepatic P450 isoforms is regulated by the gender-dependent secretory GH profiles, i.e. episodic in males and continuous in females. In the case of the feminine GH profile, the continuous presence of the hormone in the circulation completely suppresses male-specific CYP2A2, 2C11, and 3A2, while stimulating full expression of female-dependent CYP2A1, 2C7, 2C12, and non-P450 testosterone 5alpha-reductase (type 1). The gender-dependent expression of the P450s can be reversed by exposing male rats to the continuous feminine plasma GH profile and females to the episodic masculine GH profile. Under these conditions, females will now express the male-specific isoforms and suppress the female-dependent forms, whereas the opposite will occur in the males. Nevertheless, it is not clear whether the levels of expression or suppression are comparable in male and female rats exposed to the same sex-dependent GH profiles. In the present study, we have renaturalized the circulating feminine GH profile in euthyroid-maintained, hypophysectomized female and male rats at six concentrations ranging from 3-100% of normal. Continuous monitoring of GH levels revealed indistinguishable plasma profiles in females and males at each dosage administered. In the case of females, restoration of the feminine-like plasma GH profile at a concentration that was 3% of the normal level restored expression levels (i.e. mRNA, protein, and/or catalytic activity) of female-dependent CYP2C12, 2A1, and 5alpha-reductase to 50% or greater of normal and fully suppressed expression of male-specific CYP2A2, 2C11, and 3A2. Twice the dosage of the hormone (6% of normal) was required to restore female-predominant CYP2C7 to 50% of normal in hypophysectomized female rats. In contrast, we found that all of the measured isoforms were significantly less responsive to the inductive and suppressive effects of the feminine-like GH profile when administered to male rats. While suppression of the male-specific isoforms (i.e. CYP2A2, 2C11, and 3A2) in male rats required concentrations of GH in the feminine profile 2-3 times greater than were effective in female rats, no dosage of the hormone was as effective in inducing female-dependent P450s (i.e. CYP2A1, 2C7, and 2C12) in males as in females. Clearly, the continuous feminine GH profile was more effective at inducing and suppressing gender-dependent isoforms of hepatic P450 when restored to female rats, where it is normally secreted, than in males. As GH profiles appear to be the sole factor responsible for regulating the sexually dimorphic expression of hepatic P450 isoforms in adult rats, the differential responsiveness of male and female rats to the feminine GH profile are likely to be inherently induced by irreversible imprinting during a critical developmental period.