Pharmacokinetics and Postprandial Glycemic Excursions following Insulin Lispro Delivered by Intradermal Microneedle or Subcutaneous Infusion

Background

Intradermal (ID) delivery has been shown to accelerate insulin pharmacokinetics (PK). We compared the PK and pharmacodynamic (PD) effects of insulin lispro administered before two daily standardized solid mixed meals (breakfast and lunch), using microneedle-based ID or traditional subcutaneous (SC) delivery.

Method

The study included 22 subjects with type 1 diabetes in an eight-arm full crossover block design. One arm established each subject’s optimal meal dose. In six additional arms, the optimal, higher, and lower doses (+30%, -30%) were each given ID and SC delivery, in random order. The final arm assessed earlier timing for the ID optimal dose (-12 versus -2 min). The PK/PD data were collected for 6 h following meals. Intravenous basal regular insulin was given throughout, and premeal blood glucose (BG) adjusted to 115 mg/dl.

Results

The primary end point, postprandial time in range (70–180 mg/dl), showed no route-based differences with a high level of overall BG control for both SC and ID delivery. Secondary insulin PK end points showed more rapid ID availability versus SC across doses and meals (∆Tmax -16 min, ∆T50rising -7 min, ∆T50falling -30 min, all p < .05). Both intrasubject and intersubject variability for ID Tmax were significantly lower. Intradermal delivery showed modest, statistically significant secondary PD differences across doses and meals, generally within 90–120 min postprandially (∆12 mg/dl BG at 90 min, ∆7 mg/dl BGmax, ∆7 mg/dl mean BG 0–2 h, all p < .05).

Conclusions

This study indicates that ID insulin delivery is superior to SC delivery in speed of systemic availability and PK consistency and may improve postprandial glucose control.     

Thrombopoietin,but not cytokines binding to gp130 protein-coupled receptors,activates MAPKp42/44, AKT,and STAT proteins in normal human CD34+ cells,megakaryocytes, and platelets

OBJECTIVE: The development of megakaryocytes is regulated by thrombopoietin (TPO), which binds to the c-mpl receptor, and by several other cytokines such as interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), cilliary neurotropic factor (CNTF), and oncostatin (OSM), which bind to gp130 protein-coupled receptors. We attempted to identify signal transduction pathways activated by these factors in normal human megakaryocytes. MATERIALS AND METHODS: To better understand the role of these factors in normal human megakaryopoiesis we studied their effect on 1) purified human bone marrow-derived CD34+ cells, 2) human alpha(IIb)beta3+ cells (shown by immunophenotypical and morphological criteria to be megakaryoblasts), which had been expanded ex vivo from CD34+ cells in chemically defined artificial serum, and 3) gel-filtered human peripheral blood platelets. Further, in an attempt to correlate the influence of these factors on cell proliferation and survival with activation of signal transduction pathways, we evaluated their effect on the phosphorylation of MAPK p42/44 and activation of PI-3K-AKT and JAK-STAT proteins in these various cell types. RESULTS: Using serum-free liquid cultures, we found that only TPO and IL-6 protected CD34+ cells and megakaryocytes from undergoing apoptosis (decrease in annexin-V binding, PARP cleavage, and activation of caspase-3). Moreover, only TPO when used alone and IL-6 only when used in combination with TPO, stimulated the growth of human colony-forming unit-megakaryocytes (CFU-Meg) in semisolid serum-free medium. We also observed that while TPO efficiently activated various signaling pathways in CD34+ cells, megakaryocytes, and platelets (MAPK p42/44, PI-3K-AKT, STAT proteins), IL-6 stimulated phosphorylation of STAT-1, -3, and -5 proteins only in CD34+ cells and megakaryoblasts. To our surprise, none of the other gp130 protein-related cytokines tested (IL-11, LIF, CNTF, and OSM) activated these signaling pathways in CD34+ cells, megakaryoblasts, or platelets. CONCLUSIONS: Our signal transduction studies explain why TPO, by simultaneously activating several signaling pathways, is the most potent megakaryopoietic regulator and why of all five gp130 protein-related cytokines tested, only IL-6, through activation of STAT proteins, plays a role in normal human megakaryopoiesis.     

ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5. These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis. Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.

The synaptonemal complex (SC) is a proteinaceous ultrastructure that forms between homologous chromosomes (homologs) during midprophase I of meiosis and plays a critical role in coordinating the repair of programmed DNA double-strand breaks (DSBs) to form cross-over (CO) products (1, 2). At the onset of leptotene, the sister chromatids are organized into linear looped chromatin arrays conjoined at the loop bases by a protein axis that runs along the chromosomes (3, 4). Early steps in the recombination pathway enable the loose alignment of homolog axes at a distance of ∼400 nm (5). Formation of the SC then initiates and continues throughout zygotene via progressive installation of transverse filaments (TFs) that run perpendicular to the aligned homolog axes (referred to as lateral elements in the context of the SC), ultimately bringing them into close apposition along their entire length at a distance of ∼100 nm (2, 5). Installation of the TFs starts at multiple synapsis initiation sites that correspond to future Class I COs in Saccharomyces cerevisiae (6). In species with larger chromosomes such as Sordaria macrospora, synapsis initiates from CO-designated sites as well as additional sites whose distribution also appears sensitive to interference (1, 5). In Arabidopsis thaliana, 20 to 25 synapsis initiation sites per cell indicate a ∼2- to 2.5-fold excess over COs and in barley 76 synapsis initiation sites, versus 17 chiasmata reveal a ∼4.5-fold excess (7, 8). Full synapsis denotes the onset of pachytene and is maintained throughout this stage during which time CO formation is completed. As prophase I progresses to diplotene/diakinesis, the SC is disassembled.TFs have been described in a variety of organisms, and in most cases, they are composed of a single protein. These include Zip1 in budding yeast, C(3)G in Drosophila melanogaster, SYCP1 in mouse, ZYP1 in A. thaliana (encoded by duplicated genes, ZYP1a and ZYP1b), ZEP1 in rice (Oryza sativa), and ZYP1 in barley (Hordeum vulgare) (9–16). Caenorhabditis elegans is an exception that possesses six TF proteins (SYP1-6) required for normal synapsis (17–22). Despite a striking lack of homology between the TFs at the primary amino acid sequence level, they share very similar structures, comprising a globular N-terminal domain linked to another globular domain at the C terminus via a long alpha helical central region that is able to form large stretches of parallel, in-register, homodimeric coiled coils (23). Studies have shown that the TFs are oriented such that the C termini are associated with lateral elements potentially interacting with DNA, while the N-terminal domains localize to the central region (2, 24). Evidence suggests that the overall three-dimensional macromolecular organization of the SC is also somewhat conserved. Analyses in mouse, Drosophila, and H. vulgare (barley) strongly suggest that these organisms form SCs with a bilayer of TFs (25–28). A multilayered structure is also supported by studies in Blabs cribrosa (beetle) (29, 30). However, key aspects of the organization of the TFs within the SC remain a matter of debate. Initially, analysis of zip1 mutants in S. cerevisiae suggested that the TFs comprise a tetramer of two opposing Zip1 dimers with their N termini forming overlapping interactions in the central region of the SC (31). X-ray crystallographic studies of the human TF, SYCP1, report that the protein forms a tetrameric building block that self-assembles into a zipper-like lattice through “head-to-head” N-terminal interactions in the SC central region and “back-to-back” interactions between adjacent C-terminal dimers at the lateral elements (24). In contrast, analysis of the mouse SC using electron tomography has led to the proposal that the SC has a more dynamic structure with TF dimers forming a variety of less regimented interactions as part of an irregular single plane. However, this model appears inconsistent with other studies in mouse which support a more ordered structure (25, 26).Mutant analysis has demonstrated that TF proteins are essential for assembly of the SC central region and thus homolog synapsis. These also confirm an important role in the control of CO formation but with some variation between organisms. Studies of zip1 mutants in S. cerevisiae have shown that the Zip1 protein is a member of the ZMM group of proteins comprising Zip1, Zip2, Zip3, Zip4, Msh4, Msh5, and Mer3 that are required for the formation of Class I interfering COs (32). CO interference is a patterning mechanism that ensures even spacing of COs along the chromosomes (33–35). In S. cerevisiae and Arabidopsis, Class I COs account for ∼85% of total COs and the remaining Class II COs (∼15%) are randomly distributed (36–38). However, in plants, Zip1 orthologs appear to be functionally independent of the other ZMM proteins for CO formation (14–16). Genetic analysis of S. cerevisiae zip1 deletion mutants revealed a modest reduction in CO formation ∼30 to 40% with residual COs no longer exhibiting CO interference leading to the suggestion that the SC may mediate this process (10). Subsequent studies based on a molecular analysis of recombination intermediates in zip1 and other zmm mutants argue against a role for the SC in mediating interference as they indicate that the fate of DSBs is designated at an early stage in the recombination pathway prior to installation of the SC (32, 39). In female Drosophila lacking the TF protein C(3)G, DSB formation is thought to be reduced and they fail to form COs, although SC formation is independent of recombination (12). These authors also report that analysis of flies expressing a mutant version of the protein reveals that a complete SC is not required for CO interference (12). A major reduction in COs of ∼90% is also observed in mouse sycp1 mutants although DSB formation appears normal (13). Similarly in C. elegans (in which SC installation occurs at pairing centers), syp-1 and syp-2 null mutants recombination is initiated but COs do not form (17, 18). A further study in which the SC central region was partially depleted by RNA interference (RNAi)–induced SYP-1 knockdown found that CO interference was reduced leading to an increase in COs, suggesting a role for the SC in limiting COs (40).TFs have been studied in several plant species including Arabidopsis, barley, and rice (14–16). Analysis of Tos17 insertion mutants of the rice TF gene ZEP1 demonstrated that in common with other organisms, it is essential for SC formation and affects CO formation (16). However, rather than displaying a reduction in COs, analysis of the short arm of chromosome 11 revealed a more than threefold increase in COs in zep1 mutants (16). Like rice, barley is a member of the grass family (Poaceae), and in common with rice, RNAi knockdown lines of the TF protein HvZYP1 are defective in SC formation, but in contrast, CO formation is reduced to ∼25% of wild-type levels (15). In Arabidopsis, the TF protein, ZYP1, is encoded by functionally redundant duplicated genes, ZYP1a and ZYP1b, which share 93% homology and are encoded within 2 kb of each other on opposite strands of chromosome 1. Individual zyp1a and zyp1b mutants are fertile and possess only mild meiotic phenotypes, and as isolation of a double mutant has thus far proved intractable, functional analysis of ZYP1 has relied on RNAi knockdown lines (14). As expected, these lines failed to assemble an SC. Chiasma frequency was reduced by ∼20 to 30% and based on metaphase I bivalent shapes, they appeared to exhibit interference, but a proportion involved ectopic recombination with nonhomologs (14).Although existing studies imply that there may be some variation in the role of the SC in relation to CO control in plants, the studies in Arabidopsis and barley were based on RNAi knockdown lines rather than TF mutants. Hence to address this issue, we have generated CRISPR/Cas zyp1a/zyp1b mutants. This has enabled a detailed analysis of ZYP1 function in Arabidopsis, revealing that it is required for formation of the obligate CO and implementation of CO patterning. Loss of the protein also disrupts the normal program of homolog coalignment during prophase I.     

Quiescent CD34+ early erythroid progenitors are resistant to several erythropoietic 'inhibitory' cytokines; role of FLIP

In this study, quiescent bone marrow-derived CD34+ erythroid burst-forming units (BFU-E) were found to be resistant to the inhibitory effects of tumour necrosis factor (TNF)-alpha and -beta as well as interferon (IFN)-alpha, -beta and -gamma, in contrast to those stimulated by a combination of erytrhropoietin (Epo) plus kit ligand (KL). Unexpectedly, we found that TNF-alpha also inhibited the apoptosis of quiescent normal human CD34+ BFU-E cells. Accordingly, TNF-alpha added to CD34+ cells cultured for 2 d in serum-free medium protected clonogeneic BFU-E from undergoing serum deprivation-mediated apoptosis. Furthermore, the prosurvival effect of TNF-alpha in quiescent CD34+ cells was consistent with its ability to induce phosphorylation of mitogen-activated protein kinase (MAPK) p42/44. However, when added to CD34+ cells that were stimulated by Epo + KL, TNF-alpha induced apoptosis and inhibited proliferation of BFU-E. To explain this intriguing differential sensitivity between unstimulated CD34+ cells versus those stimulated by Epo + KL, we examined the expression of apoptosis-regulating genes (FLIP, BCL-2, BCL-XL, BAD and BAX) in these cells. Of all the genes tested, FLIP became rapidly downregulated in CD34+ cells 24 h after stimulation with Epo + KL, suggesting that it may protect quiescent CD34+ BFU-E progenitors residing in the bone marrow from the inhibitory effects of inflammatory cytokines. Thus, we hypothesize that cycling cells may become more sensitive to proapoptotic stimuli (e.g. chemotherapy, inhibitory cytokines) than quiescent ones because of the downregulation of protective FLIP.     

Adult patients with sporadic polycystic kidney disease: the importance of screening for mutations in the PKD1 and PKD2 genes

Background

ADPKD is one of the most common inherited disorders, with high risk for end-stage renal disease. Numerous patients, however, have no relatives in whom this disorder is known and are unsure whether they may transmit the disease to their offsprings. The aim of this study was to evaluate whether germline mutation analysis adds substantial information to clinical symptoms for diagnosis of ADPKD in these patients.

Methods

Clinical data included renal function and presence of liver or pancreas cysts, heart valve insufficiency, intracranial aneurysms, colonic diverticles, and abdominal hernias. Family history was evaluated regarding ADPKD. Germline mutation screening of the PKD1 and PKD2 genes was performed for intragenic mutations and for large deletions.

Results

A total of 324 adult patients with ADPKD including 30 patients without a family history of ADPKD (sporadic cases) were included. PKD1 mutations were found in 24/30 and PKD2 mutations in 6 patients. Liver cysts were present in 14 patients and intracranial aneurysms in 2 patients. Fourteen patients (45%) had no extrarenal involvement. Compared to the 294 patients with familial ADPKD, the clinical characteristics and the age at the start of dialysis were similar in those with sporadic ADPKD.

Conclusion

The clinical characteristics of patients with sporadic and familial ADPKD are similar, but sporadic ADPKD is often overlooked because of the absence of a family history. Molecular genetic screening for germline mutations in both PKD1 and PKD2 genes is essential for the definitive diagnosis of ADPKD.     

DSA血流定量分析软件评价血流导向装置治疗颅内大型动脉瘤的有效性

目的 探讨DSA血流定量分析软件评价血流导向装置治疗颅内大型动脉瘤后的血流特性改变,以分析血流导向装置治疗颅内大型动脉瘤的有效性.方法 回顾性分析2012年8月-2013年4月采用血流导向装置（Tubridge,Microport,上海）治疗的颅内大型动脉瘤患者15例.以相同标准采集所纳入患者治疗前后常规造影图像,并通过DSA血流定量分析软件进行图像后处理,通过生成的时间密度曲线,分析载瘤动脉远端显影延迟时间、动脉瘤瘤体内血流达峰时间、瘤体内血流曲线下面积以及瘤体内血流最大斜率的变化情况.结果 本组患者的支架置入技术成功率为100%,通过DSA分析软件术后即刻分析发现,与术前相比,载瘤动脉远端延迟时间缩短中位数（M）1.031 s（范围0.324~2.143 s）,动脉瘤瘤腔内血流曲线下面积（相对值）以及最大斜率（相对值）分别减少57±15和49±25.结论 Tubridge支架置入前后载瘤动脉远端显影时间延迟、瘤腔内血流曲线下面积及瘤腔内血流最大斜率的下降,证实采用血流定量分析方法评估血流导向装置对于颅内大型动脉瘤的即刻治疗效果是有效的,但其对于远期疗效的评估还有待进一步观察.