Effect of sitagliptin on glucose control in adult patients with Type 1 diabetes: a pilot, double-blind, randomized, crossover trial.

AIMS: Patients with Type 1 diabetes have significantly elevated postprandial glucagon secretion. Dipeptidyl peptidase IV inhibitors improve HbA(1c) by several mechanisms, including increasing glucagon-like peptide 1 and glucose-dependent insulinotropic peptide concentrations, which decreases postprandial rises in glucagon in both Type 1 and Type 2 diabetes. This study evaluates the clinical implications of sitagliptin in adult patients with Type 1 diabetes. METHODS: This investigator-initiated, double-blind, randomized, crossover, 8-week, pilot study enrolled 20 adult subjects with Type 1 diabetes. Subjects received sitagliptin 100 mg/day or placebo for 4 weeks and then crossed over. Outcomes included 2-h postprandial blood glucose and 24-h area under the curve changes in glucose measurements from continuous glucose monitoring, HbA(1c) , fructosamine and insulin dose. RESULTS: Sitagliptin significantly reduced blood glucose (2-h postprandial and 24-h area under the curve) despite reduced total and prandial insulin dose. Based on continuous glucose monitor findings, sitagliptin improved measures of glycaemic control, including mean blood glucose (-0.6 mmol/l; P = 0.012) and time in euglycaemic range 4.4-7.8 mmol/l (0.4 ± 0.2 h; P = 0.046). Significant reductions were also observed in M100, Glycemic Risk Assessment Diabetes Equation (GRADE) and J-index. After controlling for period, treatment and insulin dose, the HbA(1c) was also significantly reduced [-0.27 ± 0.11% (-2.91 ± 1.16 mmol/mol); P = 0.025] when patients were taking sitagliptin. CONCLUSIONS: Sitagliptin significantly improved overall glucose control, including postprandial and 24-h glucose control, in adult patients with Type 1 diabetes, while significantly reducing prandial insulin requirements. Further investigation is warranted in patients with Type 1 diabetes in a larger cohort designed to assess both clinical outcomes and mechanism of action.

c-Myb down-regulation is associated with human colon cell differentiation, apoptosis, and decreased Bcl-2 expression.

c-myb is expressed in human and murine colonic mucosa and elevated expression occurs in premalignant adenomatous polyps and carcinomas. c-Myb is required for colon cell proliferation, and there is evidence of c-myb down-regulation during differentiation. Recently, c-myb has been implicated in hematopoietic cell survival via regulation of bcl-2 gene expression. However, c-myb expression during terminal differentiation and apoptosis in the colonic crypt has not been examined. The experiments in this study examine the spatial and temporal expression of c-Myb protein in vivo using human colonic crypt sections and in vitro in human colon tumor cell lines undergoing butyrate-induced differentiation and apoptosis. Electron microscopy, together with molecular and biochemical analysis, was used to define the differentiation status of the cells. Results demonstrate a decrease in c-Myb expression during the commitment of cells to differentiation and apoptosis. Decreased levels of c-Myb are accompanied by a decrease in Bcl-2. These data suggest that the transcription factor c-Myb has a role in regulating the balance between proliferation, differentiation, and apoptosis in the colonic crypt. Furthermore, elevated c-Myb levels in colon tumor cells may lead to persistent bcl-2 expression, thus protecting tumor cells from programmed cell death.

Megakaryocyte maturation in long-term marrow culture.

Evidence has been sought for megakaryocyte maturation in long-term cultures of mouse bone marrow. Cultures up to 14 weeks of age were examined for the presence of megakaryocytes with processes, that is, resembling the morphological appearance seen in vivo prior to platelet liberation. Such cells were found floating just above the adherent stromal layer using low magnification phase contrast microscopy. It was rare to observe as many as 20 of these cells per 25-cm2 flask. At higher magnification, processes were seen to be attenuated with constrictions at intervals along their length. Time-lapse photography was used to follow the development and behavior of the processes. Direct evidence of rupture was very rare; generally the megakaryocytes retracted their processes within 48 h. Careful searching of cultures occasionally revealed the presence of several process fragments, and sometimes individual platelets were found. Ultrastructurally, the processes were seen to contain organelles that are usually associated with platelets. The observations applied to both Dexter and Whitlock-Witte cultures. It is concluded that maturation of megakaryocytes occurs in long-term marrow culture to the point where platelet release appears imminent. Final rupture is rare and may require shearing forces, which in vivo would be provided by blood flow.

Two tandemly arrayed genes encode the (histidine) secretory acid phosphatases of Leishmania donovani.

Leishmania donovani promastigotes constitutively secrete a glycosylated and phosphorylated acid phosphatase activity. This secretory acid phosphatase (SAcP) was purified from L. donovani culture supernatants and amino-acid sequence was obtained from both the N-terminus and a tryptic peptide fragment derived from the isolated protein. A polymerase chain reaction (PCR)-based strategy, using degenerate oligo primers designed from the amino-acid sequence data, identified two single-copy, tandemly arrayed open reading frames (ORFs) capable of encoding the L. donovani SAcP (SAcP-1, 2052 bp and SAcP-2, 2124 bp). Both SAcP-1 and -2 were shown to be actively transcribed by L. donovani promastigotes by reverse transcription (RT) and PCR amplification. The deduced amino-acid sequences of SAcP-1 and SAcP-2 show high conservation to each other in four regions: a 23-amino-acid signal peptide; a catalytic domain containing several potential N-linked glycosylation sites; a Ser/Thr-rich repeat region containing multiple potential phosphorylation sites and a common C-terminus. Within the catalytic domain, the L. donovani SAcPs possess two conserved consensus sequences characteristic of histidine acid phosphatases (AcPs). Furthermore, antisera to native L. donovani SAcP immunoprecipitated in vitro transcription/translation products of both SAcP-1 and SAcP-2. Cumulatively, these data indicate that the acid phosphatase activity constitutively secreted by L. donovani promastigotes is composed of two (histidine) AcP isoforms that are encoded by SAcP-1 and SAcP-2, respectively.

Programmed cell death in extraocular muscle tendon/sclera precursors.

PURPOSE: This study was designed to examine the occurrence of natural cell death in the periocular mesenchyme of mouse embryos. METHODS: Vital staining with LysoTracker Red and Nile blue sulfate as well as terminal nick end labeling (TUNEL) were utilized to identify apoptotic cell death in whole and histologicaly sectioned gestational day 10.5 to 14 mouse embryos. Laser scanning confocal microscopy was used to provide a three dimensional representation of the cell death pattern. Immunohistochemical staining for neural crest and myoblast populations was utilized to indicate the cell population undergoing apoptosis. RESULTS: Programmed cell death was evident in the developing rectus muscle tendons/sclera on gestational days 11 through 12.5 (corresponding to the weeks 5-6 of human development). Although each of these peripheral periocular condensations has readily apparent amounts of apoptosis, the pattern of cell death varied among them. Cell death was most apparent in the superior rectus tendon primordium, while that for the lateral rectus had the least evidence of apoptosis. CONCLUSIONS: Although apoptosis was readily evident in the periocular mesenchyme in distinct regions located medial and distal to the developing rectus muscles, programmed cell death in these sites has not previously been reported. New imaging techniques coupled with stains that evidence apoptotic cell death have made it possible to define this tissue as a prominent region of programmed cell death. Although neuronal tissues, including particular regions of the developing eye, are well recognized as sites of programmed cell death, description of this phenomenon in the extraocular tendon/sclera precursors is novel.

Reduced mononuclear leukocyte ascorbic acid content in adults with insulin-dependent diabetes mellitus consuming adequate dietary vitamin C.

Several recent studies suggest that vitamin C (ascorbic acid [AA]) status may be altered in insulin-dependent diabetes mellitus (IDDM). We measured the AA content of mononuclear leukocytes (MN-AA) as an indicator of tissue vitamin C status in adults with IDDM and nondiabetic adults matched for age and sex. Dietary vitamin C intake and plasma AA were analyzed to ensure that vitamin C availability was adequate. Dietary vitamin C intakes were above recommendations and were not different between the groups. MN-AA was reduced by 33% on average (P less than .05) in adults with IDDM (1.75 microgram/mg total protein [TP]) when compared with nondiabetics (2.60 micrograms/mg TP). When MN-AA is indexed to the dietary vitamin C intake (MN-AA/100 mg diet C), the storage deficit in adults with IDDM averages 50% (P less than .05). This observation suggests an impaired tissue AA storage in adults with IDDM and supports the theory that intracellular scurvy contributes to the chronic degenerative complications of the disease.

Types of interventions made by clinical pharmacists in the IMPROVE study. Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers.

The purpose of this study was to describe and evaluate the activities and interventions provided by ambulatory care clinical pharmacists during the IMPROVE (Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers) study. A total of 523 patients were randomized into the intervention arm at nine Veterans Affairs medical centers if they were considered to be at high risk for drug-related problems. Patients randomized to the control group had no interventions and they are not reported. Using a standard form, pharmacists were asked to document the length of visit, method of contact, medical conditions addressed, and drug-related problems addressed and resolved during each contact. Seventy-eight ambulatory care clinical pharmacists documented 1855 contacts over 12 months, an average of 3.54 +/- 2.31/patient. The length of visits was 15 minutes or more for 73% of contacts. In-person contacts accounted for 1421 visits (76.6%), with the remainder being telephone contacts. During each contact the average number of drug-related problems addressed and resolved were 1.64 +/- 1.16 and 1.14 +/- 0.98, respectively. More drug-related problems were addressed and resolved when visits were 15 minutes or longer (p=0.001) and when the contact was in person (p=0.001). These data may provide information to clinical pharmacists developing pharmacy-managed clinics for patients at high risk for drug-related problems. The information may be a benchmark for types of interventions that can be made, as well as the time commitments required to make them.

Clinical and economic impact of ambulatory care clinical pharmacists in management of dyslipidemia in older adults: the IMPROVE study. Impact of Managed Pharmaceutical Care on Resource Utilization and Outcomes in Veterans Affairs Medical Centers.

We examined the impact of ambulatory care clinical pharmacist interventions on clinical and economic outcomes of 208 patients with dyslipidemia and 229 controls treated at nine Veterans Affairs medical centers. This was a randomized, controlled trial involving patients at high risk of drug-related problems. Only those with dyslipidemia are reported here. In addition to usual medical care, clinical pharmacists were responsible for providing pharmaceutical care for patients in the intervention group. The control group did not receive pharmaceutical care. Seventy-two percent of the intervention group and 70% of controls required secondary prevention according to the National Cholesterol Education Program guidelines. Significantly more patients in the intervention group had a fasting lipid profile compared with controls (p=0.021). The absolute change in total cholesterol (17.7 vs 7.4 mg/dl, p=0.028) and low-density lipoprotein (23.4 vs 12.8 mg/dl, p=0.042) was greater in the intervention than in the control group. There were no differences in patients achieving goal lipid values or in overall costs despite increased visits to pharmacists. Ambulatory care clinical pharmacists can significantly improve dyslipidemia in a practice setting designed to manage many medical and drug-related problems.

Radiation response of mouse lymphoid and myeloid cell lines. Part II. Apoptotic death is shown by all lines examined.

The mode of death induced by gamma-irradiation in a panel of 10 mouse lymphoid or myeloid cell lines was examined. Four of these lines were known to lose viability (membrane integrity) rapidly after irradiation, whilst the others were known to lose viability considerably more slowly. However, based on the criteria of morphology and DNA degradation pattern, all 10 lines showed apoptotic death. The occurrence of apoptosis after irradiation in rapid-dying lymphoid cell lines was consistent with published results, whilst the demonstration of apoptosis in slow-dying lines was unexpected. Cells of the slow-dying lymphoid lines underwent one or more mitoses prior to death, a feature also reported for fibroblastoid cell lines. However, the occurrence of radiation-induced necrosis in fibroblasts suggests that the pathways leading to 'mitotic death' differ between fibroblastoid and lymphoid cell lines.

A practical method to measure GFR in people with type 1 diabetes.

AIMS: Improved early diagnostic methods are needed to identify risk for kidney disease in people with type 1 diabetes. We hypothesized that glomerular filtration rate (GFR) measured by iohexol clearance in dried blood spots (DBS) on filter paper would be comparable to plasma (gold-standard) and superior to estimated GFR (eGFR) and, second, that adjustment for ambient blood glucose would improve accuracy and precision of GFR measurement. METHODS: GFR was measured by iohexol clearance in plasma, DBS, and as estimated by the CKD-Epidemiology Collaboration equations in 15 adults with type 1 diabetes at two visits, one euglycemic and one hyperglycemic. RESULTS: GFR measured by DBS was more comparable and less biased than GFR cystatin C, serum creatinine, and both combined. GFR was higher during hyperglycemia. Correction for between visit glycemia statistically significantly reduced bias and mean squared error for GFR measured by DBS as compared to gold-standard during euglycemia. CONCLUSIONS: Iohexol clearance measured with DBS performed better than eGFR methods. Correction for ambient blood glucose improved precision and accuracy of GFR measurement. This method is more convenient than the gold-standard GFR method and may improve screening and diagnostic capabilities in people with type 1 diabetes, especially when GFR is >60ml/min/1.73m(2).

Leishmania: amastigotes synthesize conserved secretory acid phosphatases during human infection.

Leishmania donovani is the major causative agent of Old World human visceral leishmaniasis (VL). In vitro, both promastigotes and axenic amastigotes of L. donovani constitutively secrete soluble acid phosphatases (SAcPs), which contain conserved antigenic epitopes. These SAcPs are the most abundant and best characterized secretory proteins of this parasite. The aim of this study was to determine whether this enzyme was produced by intracellular amastigotes during the course of human infection. To that end, sera from acutely infected leishmaniasis patients were tested for anti-SAcP antibodies using L. donovani promastigote culture supernatants. Our results showed that VL patient sera from different endemic foci immunoprecipitated parasite SAcP enzyme activity. Further, these VL patient sera recognized the 110- and 130-kDa SAcPs in both Western blots and radioimmunoprecipitation assays. Results of tunicamycin experiments demonstrated that VL patient anti-SAcP antibodies were directed against the polypeptide backbone of the parasite SAcPs. In addition, both radiolabeled L. donovani SAcPs and native enzyme activities were immunoprecipitated by sera from patients with various forms of cutaneous leishmaniasis. Together, these studies demonstrate that Leishmania amastigotes produce SAcPs during the course of human infections.

Bell's palsy in an older patient with uncontrolled hypertension due to medication nonadherence.

OBJECTIVE: To describe and inform pharmacists of a rarely reported occurrence of facial palsy in an elderly patient with uncontrolled hypertension resulting from nonadherence to blood pressure medications. CASE SUMMARY: A 62-year-old Hispanic woman presented to the hypertension clinic with left facial weakness, mild eyelid lag, and auricular pain for two days. The patient self-discontinued fosinopril and minoxidil six days and two days prior to developing these symptoms, respectively. A diagnosis of idiopathic peripheral VII cranial nerve lesion was made after ruling out other possible causes. Corticosteroids were not initiated because of this patient's labile hypertension. Palliative therapy was initiated and the left facial paralysis continuously improved during the six months after discharge. DISCUSSION: Patients have rarely presented with facial paralysis as the initial feature of severe hypertension. The relationship between facial paralysis and hypertension has been reported in a small number of cases, including several reports of recurrence of paralysis during acute exacerbations of hypertension. A variety of physiologic theories to explain the relationship between facial paralysis and hypertension have been published, including small hemorrhages into the facial canal which have been confirmed by two autopsies. However, the true etiology remains unknown. CONCLUSIONS: The possible relationship between facial paralysis and uncontrolled hypertension has not been reported in pharmacy literature and has been reported only twice in subspecialty medical journals since 1990. Pharmacists should be aware of the complications of hypertension and should question patients about signs and symptoms at each visit. While Bell's palsy complicating hypertension does not appear to be a serious complication, pharmacists must appreciate that the patient should be immediately evaluated to rule out a more serious neurologic event.

Preferential pre-mRNA utilisation of an upstream cryptic 5' splice site created by a single base deletion mutation in exon 37 of the FBN-1 gene.

A heterozygous deletion of a single base (A4704) from exon 37 of the fibrillin-1 gene was defined in a patient with Marfan syndrome and subsequently in his previously undiagnosed father. The deletion created a cryptic 5' splice site in exon 37 which was utilised in preference to the normal 5' splice site during pre-mRNA processing in skin fibroblasts cultured from the proband. The mutant mRNA showed a 48-bp deletion from the 3' end of exon 37 which was predicted to restore the reading frame in the mutant mRNA and result in the deletion of a 16-amino-acid sequence from a central eight-cysteine repeat motif of the fibrillin-1 molecule. Interestingly, the cryptic 5' splice site in exon 37 and the normal 5' splice site had equally strong consensuses for splice-site selection. The preferential utilisation of the cryptic site is discussed in relation to current theories on the mechanisms involved in pre-mRNA splicing. Analysis by reverse-transcription PCR indicated that, in the patients skin fibroblasts, the steady-state level of the mis-spliced mutant mRNA was close to that from the normal allele. In addition, evidence from immunoblotting and pulse-chase biosynthetic labelling indicated that close to normal amounts of fibrillin-1 were being synthesised and secreted by the cells. However, in contrast to control cells cultured from an unaffected individual, little fibrillin-1 was detected, either biosynthetically or by immunofluorescence, in the extracellular matrix produced by the proband's fibroblasts. Thus, the slightly shorter mutant fibrillin-1 molecules appeared to be exerting a powerful dominant-negative effect on the incorporation of normal fibrillin-1 molecules into microfibrils in this culture system. This severe inhibition of microfibril synthesis in cell culture contrasts with the 'classic' phenotype of the proband, suggesting that factors influencing microfibril formation may differ greatly between in vivo and in vitro environments.

Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion. Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p. Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P(2), forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain. Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection. Triple null mutant 5-phosphatase strains are non-viable. To investigate the role of PtdIns(4,5)P(2) in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P(2) 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P(2) levels to near basal values. Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains. Cellular PtdIns(4,5)P(2) levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for Deltainp51Deltainp53, Deltainp51Deltainp52 and Deltainp52Deltainp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression. Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P(2) accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P(2) 5-phosphatases.