Secretory Component Research Articles

High prevalence of IgA class anti-neutrophil cytoplasmic antibodies (ANCA) is associated with increased risk of bacterial infection in patients with cirrhosis

Anti-neutrophil cytoplasmic antibodies (ANCA) are a non-uniform family of antibodies recognizing diverse components of neutrophil granulocytes. ANCA formation might be induced by protracted bacterial infections or probably reflect an abnormal immune response to commensal microorganisms. Bacterial infections are common complications in cirrhosis with high incidence of episodes caused by enteric organisms, therefore, we sought to study the presence and clinical importance of ANCA in cirrhosis. Sera of 385 patients with cirrhosis of different etiologies were assayed for ANCA of IgG, IgA, IgA1, IgA2, and secretory IgA subtypes by indirect immunofluorescence and ELISAs. The control group comprised 202 patients with chronic liver diseases without cirrhosis and 100 healthy subjects. In cirrhosis, a 2-year follow-up, observational study was conducted to assess a possible association between the presence of ANCA and clinically significant bacterial infections. Prevalence of ANCA IgA was significantly higher in cirrhosis (52.2%) compared to chronic liver diseases (18.6%) or healthy controls (0%, p<0.001 for both). ANCA IgA subtyping assays revealed marked increase in the proportion of IgA2 subtype (46% of total ANCA IgA) and presence of the secretory component concurrently. Presence of ANCA IgA was associated with disease-specific clinical characteristics (Child-Pugh stage and presence of ascites, p<0.001). During a 2-year follow-up period, risk of infections was higher among patients with ANCA IgA compared to those without (41.8% vs. 23.4%, p<0.001). ANCA IgA positivity was associated with a shorter time to the first infectious complication (pLogRank <0.001) in Kaplan-Meier analysis and was identified as an independent predictor in multivariate Cox-regression analysis (HR:1.74, 95% CI: 1.18-2.56, p=0.006). Presence of IgA type ANCA is common in cirrhosis. Involvement of gut mucosal immune system is in center of their formation and probably reflects sustained exposure to bacterial constituents.

Polymeric immunoglobulin receptor traffics through two distinct apically targeted pathways in primary lacrimal gland acinar cells

The polymeric immunoglobulin receptor (pIgR) mediates transcytosis of dimeric immunoglobulin A (dIgA) and its release into mucosal secretions. The present study reveals the complexity of the trafficking of pIgR to the apical plasma membrane in epithelial cells with exocrine secretory functions; in rabbit lacrimal gland acinar cells (LGACs), trafficking of pIgR involves both the transcytotic pathway and one arm of the regulated secretory pathway. By specifically tracking pIgR endocytosed from the basolateral membrane, we show here that the Rab11a-regulated transcytotic pathway mediates the basal-to-apical transport of pIgR, and that pIgR sorted into the transcytotic pathway does not access the regulated secretory pathway. However, previous work in LGACs expanded in the present study has shown that some pIgR is localized to Rab3D-enriched mature secretory vesicles (SVs). Myosin Vb and myosin Vc motors modulate release of proteins from the Rab11a-regulated transcytotic pathway and the Rab3D-enriched secretory pathway in LGACs, respectively. Confocal fluorescence microscopy and biochemical assays showed that inhibition of myosin Vb and myosin Vc activity by overexpression of their dominant-negative mutants each significantly but differentially impaired aspects of apically targeted pIgR trafficking and secretory component release, suggesting that these motors function to regulate pIgR trafficking in both the transcytotic and exocytotic pathways. Intriguingly, a second mature SV population enriched in Rab27b was devoid of pIgR cargo, suggesting the specialization of Rab3D-enriched mature SVs to carry a particular subset of cargo proteins from the trans-Golgi network to the apical plasma membrane.

Abstract 4764: The pathogenesis of ovarian Brenner tumors.

Abstract Benign Brenner tumors (BTs) are composed of nests of epithelial cells resembling urothelium, which are surrounded by a dense fibromatous stroma and are considered the benign counterpart of ovarian transitional cell tumors. Their origin remains enigmatic. We have previously described the junction between the fallopian tubal epithelium and the mesothelium of the tubal serosa, and reported that transitional cell metaplasia (TM) is frequently found in this location. We hypothesize that BTs arise from TM epithelium that, when embedded in the ovary as Walthard cell nests (WCs), may develop into BTs, some of which may progress to so-called “atypical proliferative BTs”. The aim of the present study was to evaluate the immunohistochemical (IHC) and molecular genetic features of BTs in an attempt to confirm this hypothesis. A total of 17 ovaries, 25 peritoneal mesothelia, 38 fallopian tubes, 10 TMs, 56 WCs, 48 BTs and 7 atypical proliferative BTs were included in this study. IHC for AKR1C3, alpha-inhibin (INH), AR, β-catenin, calretinin (CRT), CEA, CYP19, ER, p16, PAX2, PAX8, PR, SF1, and WT1, and fluorescence in situ hybridization (FISH) for CDKN2A (p16 encoding gene) were performed. Genomic DNA from 20 BTs was extracted for sequence analysis of BRAF, CTNNB1, ERBB2, FOXL2, KRAS, PIK3CA, and PPP2R1A mutations. A somatic mutation of PIK3CA was detected in the stromal component in one BT. No other mutations were detected in either the epithelial or the stromal component in the remaining BTs. IHC analysis revealed that fallopian tubal secretory cells, TM, WC and epithelial component of BTs and atypical proliferative BTs shared the same IHC profile, consistently expressing AKR1C3, AR, but not CRT, as opposed to mesothelium and ovarian surface epithelium. The stromal component of the tumors showed weak expression of AKR1C3 in 23/26 (88%), AR in 26/29 (90%), ER in 32/42 (74%), PR in 38/43 (88%), and WT1 in 13/41 (32%), and strong expression of CRT in 34/41 (83%), INH in 33/41 (80%), and SF1 in 38/40 (95%) BTs. Notably, CRT, INH, and SF1 showed particularly strong staining in the stromal cells that immediately surrounded the epithelial nests. In contrast, expression of these biomarkers was absent in the BT epithelium. p16 was intensely and diffusely positive in the epithelial component of 9/13 (69%), patchy in 3/13 (23%), and negative in 1/13 (8%) benign BTs. In contrast all 7 atypical proliferative BTs were completely negative for p16 in the epithelial component. FISH identified homozygous deletion of CDNK2A in all atypical proliferative BTs, but CDKN2A was retained in benign BTs (Fisher exact test two sided p-value&amp;lt;0.0001). In summary, our findings suggest: 1) tubal origin of BTs through TM and WC, based on similar IHC profile, 2) an active role of the ovarian stroma in the pathogenesis of BT, based on the periepithelial IHC pattern of CRT, INH, and SF1, markers of steroidogenic cells, along with the epithelial expression of AR, 3) the role of CDKN2A loss in the progression of BT to atypical proliferative BT. Citation Format: Elisabetta Kuhn, Ayse Ayhan Ayhan, Jeffrey D. Seidman, Robert J. Kurman. The pathogenesis of ovarian Brenner tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4764. doi:10.1158/1538-7445.AM2013-4764 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Effect of Hyperoxia on Pulmonary SIgA and Its Components, IgA and SC

PurposeOxygen therapy (hyperoxia) is essential for the treatment of some neonatal critical care conditions. The lung is the primary target for the changes induced by hyperoxia. Secretory immunoglobulin A (SIgA), IgA and secretory component (SC) reflect the local immunity in the respiratory tract induced by hyperoxia.MethodsThe enzyme-linked immunosorbent assay, immunohistochemistry staining, Western blot and Real-time PCR were used to detect the levels of cytokines, IgA and SIgA in bronchoalveolar lavage as well as IgA and SC/pIgR in pulmonary tissue.ResultsThe levels of IgA and SIgA in BAL fluid were gradually increased following neonatal rat development. Compared with air-inhaling group, in the hyperoxia group IgA, SIgA and other cytokines except IL-1 in BAL fluid were significantly elevated on the 3rd, 5th and 7th days, but on the 10th day TNF-α, SIgA and IgA rapidly decreased. In the hyperoxia group, both the protein expression of SC/pIgR and the mRNA expression of SC/pIgR were remarkably increased on the 3rd, 5th and 7th days, but were significantly decreased on the 10th day, respectively. Conclusion: The large amount of SIgA, IgA and SC in the early period of hyperoxia might protect the lungs of the neonatal rats against acute pulmonary injury, however, in the late period of hyperoxia, the abruptly drop of SIgA and its component might lead to pulmonary immunity abnormality. In hyperoxia, the increased expression of cytokines might contribute to the expression of IgA and SC.

TNF-α Mediated Secretory Component is Positively Regulated by PI3K/AKT/MTOR in CACO-2 Cells

Summary Background: Secretory component (SC) is the extracellular component of the polymeric immunoglobulin receptor (pIgR) that functions as a receptor for externally secreted polymeric immunoglobulins such as secretory IgA. SC expression is regulated by a signaling pathway involving TNF-a. Methods: Caco-2 cells were cotreated with TNF-a plus either Wortmannin, which inhibits PI3K, or Rapamycin, which inhibits mTOR. The expression of SC and pIgR mRNA were assessed by immunocytochemistry, Western blotting, and Quantitative real-time PCR. Results: Wortmannin and Rapamycin decreased the fraction of cells expressing SC, as well as the total expression of SC protein and pIgR mRNA. Conclusions: TNF-a regulation of SC expression is mediated through a PI3K/AKT/mTOR signaling pathway in Caco-2 cells.

Effect of host defenses on Clostridium difficile toxin–induced intestinal barrier injury

The severity of Clostridium difficile-associated infection depends on the virulence factors of the organism and host factors, including intestinal barrier function. The intestinal mucus layer has recently been recognized as the first line of defense against enteric pathogens. Its interaction with mucosal humoral immunity provided by secretory immunoglobulin A (SIgA) is unknown as it relates to C. difficile disease severity. This was studied in vitro. Confluent HT29 (non-mucus-producing) and HT29-MTX (mucus-producing) intestinal epithelial cells (IECs) with and without transcytosed SIgA were exposed to C. difficile toxin A (6 hours), and IEC toxin internalization, permeability (fluorescein isothiocyanate dextran), and necrosis (propidium iodide staining) were determined. In other experiments, colostral SIgA was added to the apical surface of IEC, and cleavage was determined by measurement of intact SIgA and secretory component fractions by enzyme-linked immunosorbent assay. Tumor necrosis factor α and interleukin 6 were measured from basal chamber culture supernatants by enzyme-linked immunosorbent assay. Toxin A uptake and subsequent enterotoxic effects on IEC were decreased by both the mucus layer and SIgA. Similar findings were noted with the effects of toxin A on IEC monolayer permeability, cytoskeleton changes, and proinflammatory cytokine release. The combination of the mucus layer and SIgA afforded the best protection against the adverse effects on IEC by toxin A. It seems that the mucus layer was also protective against SIgA cleavage, resulting in reduced protease activity by HT29 cells exposed to toxin A. Both intestinal mucus and SIgA were important in limiting C. difficile-associated disease severity in this model. A synergistic effect of mucus and IgA was also noted and may be due to protection of SIgA from proteolytic cleavage.

Metalloproteomic approach to the determination of calcium, iron and zinc bound to secretory immunoglobulin A in human milk

At this present work, the presence of calcium, iron and zinc bound to secretory immunoglobulin A (sIgA) from human milk was investigated. The sIgA components were separated by two‐dimensional electrophoresis (2D‐PAGE) and they were identified by electrospray ionization mass spectrometry (ESI/MS/MS). A train of eight spots with 5.4&lt;pI&lt;6.5 and a mass of 78 kDa were identified as the Ig alpha‐1 chain constant region. Another spot, with pI of 5.8 and a mass of 100 kDa was identified as transmembrane secretory component, also known as the polymeric immunoglobulin receptor (pIgR). The metal ions in the spots were detected by flame atomic absorption spectrometry (FAAS) after acid mineralization of the spots. It was discovered calcium bound to both pIgR and Ig alpha‐1 constant region, while zinc was found to be bound to the Ig alpha‐1 constant region. The association of a metal ion to a protein (subject of study of metalloproteomics) is important for many reasons and the discoveries of this study can lead to a better understanding of mechanisms of action and additional roles that sIgA and its components have in human milk. Financial support: FAPESP (2011/03291–0).

Combinatorial Analysis of Secretory Immunoglobulin A (sIgA) Expression in Plants

Delivery of secretory immunoglobulin A (sIgA) to mucosal surfaces as a passive immunotherapy agent is a promising strategy to prevent infectious diseases. Recombinant sIgA production in plants requires the co-expression of four transcriptional units encoding the light chain (LC), heavy chain (HC), joining chain (JC) and secretory component (SC). As a way to optimize sIgA production in plants, we tested the combinatorial expression of 16 versions of a human sIgA against the VP8* rotavirus antigen in Nicotiana benthamiana, using the recently developed GoldenBraid multigene assembly system. Each sIgA version was obtained by combining one of the two types of HC (α1 and α2) with one of the two LC types (k and λ) and linking or not a KDEL peptide to the HC and/or SC. From the analysis of the anti-VP8* activity, it was concluded that those sIgA versions carrying HCα1 and LCλ provided the highest yields. Moreover, ER retention significantly increased antibody production, particularly when the KDEL signal was linked to the SC. Maximum expression levels of 32.5 μg IgA/g fresh weight (FW) were obtained in the best performing combination, with an estimated 33% of it in the form of a secretory complex.

B and T cell epitope mapping and study the humoral and cell mediated immune response to B–T constructs of YscF antigen of Yersinia pestis

YscF antigen, a type III secretion protein has recently been shown partial protection in murine model. Five peptides of YscF antigen were predicted using DNASTAR and T-cell prediction software. Peptides were synthesised and authenticated using competitive, direct binding immunoassay with anti YscF/peptide sera raised in mice. Peptide P1 and P2 were found to be B cell epitope while P3 was minor B cell epitope. P4 peptide was a pure T cell epitope based on lymphoproliferative response, cytokines profile and T-bet expression. Furthermore, with an intention to enhance immunogenicity, three B-T constructs were designed between the above epitopes. Conjugate B1T1 and B2T1 showed higher serum IgG/IgA titre, respectively, as well as high secretory IgA plus secretory component (Sc) both in lung and intestinal washes. Also, these conjugates showed high T-cell proliferation in addition to higher Th1 type cytokines (IFN-γ and IL-2) in cells obtained from spleen, lamina propria and Peyer's patches. B3T1 stimulated cells showed moderate levels of IFN-γ and IL-2 but higher levels of IL-4. This study demonstrates superior immunogen of B1T1 and B2T1 of YscF antigen to be exploited as vaccine candidate for plague.

Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus

Proteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1(gp-340), mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen.

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

Secretory IgA: Designed for Anti-Microbial Defense.

Prevention of infections by vaccination remains a compelling goal to improve public health. Mucosal vaccines would make immunization procedures easier, be better suited for mass administration, and most efficiently induce immune exclusion – a term coined for non-inflammatory antibody shielding of internal body surfaces, mediated principally by secretory immunoglobulin A (SIgA). The exported antibodies are polymeric, mainly IgA dimers (pIgA), produced by local plasma cells (PCs) stimulated by antigens that target the mucose. SIgA was early shown to be complexed with an epithelial glycoprotein – the secretory component (SC). A common SC-dependent transport mechanism for pIgA and pentameric IgM was then proposed, implying that membrane SC acts as a receptor, now usually called the polymeric Ig receptor (pIgR). From the basolateral surface, pIg-pIgR complexes are taken up by endocytosis and then extruded into the lumen after apical cleavage of the receptor – bound SC having stabilizing and innate functions in the secretory antibodies. Mice deficient for pIgR show that this is the only receptor responsible for epithelial export of IgA and IgM. These knockout mice show a variety of defects in their mucosal defense and changes in their intestinal microbiota. In the gut, induction of B-cells occurs in gut-associated lymphoid tissue, particularly the Peyer’s patches and isolated lymphoid follicles, but also in mesenteric lymph nodes. PC differentiation is accomplished in the lamina propria to which the activated memory/effector B-cells home. The airways also receive such cells from nasopharynx-associated lymphoid tissue but by different homing receptors. This compartmentalization is a challenge for mucosal vaccination, as are the mechanisms used by the mucosal immune system to discriminate between commensal symbionts (mutualism), pathobionts, and overt pathogens (elimination).

Stress modulates intestinal secretory immunoglobulin A

Stress is a response of the central nervous system to environmental stimuli perceived as a threat to homeostasis. The stress response triggers the generation of neurotransmitters and hormones from the hypothalamus pituitary adrenal axis, sympathetic axis and brain gut axis, and in this way modulates the intestinal immune system. The effects of psychological stress on intestinal immunity have been investigated mostly with the restraint/immobilization rodent model, resulting in an up or down modulation of SIgA levels depending on the intensity and time of exposure to stress. SIgA is a protein complex formed by dimeric (dIgA) or polymeric IgA (pIgA) and the secretory component (SC), a peptide derived from the polymeric immunoglobulin receptor (pIgR). The latter receptor is a transmembrane protein expressed on the basolateral side of gut epithelial cells, where it uptakes dIgA or pIgA released by plasma cells in the lamina propria. As a result, the IgA-pIgR complex is formed and transported by vesicles to the apical side of epithelial cells. pIgR is then cleaved to release SIgA into the luminal secretions of gut. Down modulation of SIgA associated with stress can have negative repercussions on intestinal function and integrity. This can take the form of increased adhesion of pathogenic agents to the intestinal epithelium and/or an altered balance of inflammation leading to greater intestinal permeability. Most studies on the molecular and biochemical mechanisms involved in the stress response have focused on systemic immunity. The present review analyzes the impact of stress (mostly by restraint/immobilization, but also with mention of other models) on the generation of SIgA, pIgR and other humoral and cellular components involved in the intestinal immune response. Insights into these mechanisms could lead to better therapies for protecting against pathogenic agents and avoiding epithelial tissue damage by modulating intestinal inflammation.

An attempt to identify antigen for IgA pemphigus accidentally showed the presence of transmembrane secretory component in keratinocytes

In this study, to identify unknown autoantigen for intraepidermal neutrophilic IgA dermatosis type IgA pemphigus (IEN), we performed a new immunoprecipitation (IP), in which biotinylated KU-8 cell extract was precipitated by peptide M agarose, that has specific binding ability to human IgA. By the IP combined with mass spectrometry, we identified transmembrane secretory component (TSC) as a candidate. TSC was attractive as an antigen for IEN, because TSC delivers IgA in the mucosae. Polyclonal antibody to TSC reacted with the epidermis in immunofluorescence (IF).

Human Milk sIgA Molecules Contain Various Combinations of Different Antigen-Binding Sites Resulting in a Multiple Binding Specificity of Antibodies and Enzymatic Activities of Abzymes

In the classic paradigm, immunoglobulins are monospecific molecules that have stable structures and two or more identical antigen-binding sites. However, we show here for the first time that the sIgA pool of human milk contains, depending on the donor, only 35±5% λ-sIgAs, 48±7% κ-sIgAs, and 17±4% of chimeric λ-κ-sIgAs. sIgA preparations contained no traces of canonical enzymes. However, all sIgA fractions eluted from several specific affinity sorbents under the conditions destroying even strong immune complexes demonstrated high catalytic activities in hydrolysis of ATP, DNA, and oligosaccharides, and phosphorylation of proteins, lipids, and oligosaccharides. Sequential re-chromatographies of the sIgA fractions with high affinity to one affinity sorbents on the second, third and then fourth affinity sorbents bearing other immobilized antigens led to the distribution of Abs and all catalytic activities all over the profiles of these chromatographies; in all cases some fractions eluted from affinity sorbents only under the conditions destroying strong immune complexes. In vitro, only an addition of reduced glutathione and milk plasma containing no Abs to two sIgA fractions with different affinity for DNA-cellulose led to a transition of up to 11–20% of Ab from one fraction to the other. Our data are indicative of the possibility of half-molecule exchange between different IgA and sIgA molecules. In addition, it cannot be excluded that during the penetration of IgAs through the specific milk barrier, the secretory component (S) and the join chain (J) can combine molecules of dimeric H2L2 λ-IgAs and κ-IgAs against different antigens forming many different variants of H4L4SJ sIgA molecules. Therefore, some chimeric molecules of sIgA can contain from two to four HL-fragments to various antigens interacting with high affinity with different sorbents and catalyzing various chemical reactions. Our data essentially expand the ideas concerning explanation of the phenomenon of polyspecificity and cross-reactivity of Abs.

Investigation of the introduction of three times daily injections of Insulin Lispro Mixture‐50 on an outpatient basis: Therapeutic effects of 12 months’ treatment with and without concomitant sulfonylurea

There is no definitive evidence as to whether oral hypoglycemic agents (OHA) should be continued or withdrawn prior to the introduction of insulin therapy on an outpatient basis. Insulin Lispro Mixture-50 (LM-50), which supplements basal insulin secretion because of the presence of the intermediate-acting component contained in it, may enable more stable glucose control and allow discontinuation of prolonged OHA therapy, such as with sulfonylureas (SU). To determine whether the introduction of three times daily (t.i.d.) injections of LM-50 on an outpatient basis was effective in yielding adequate glucose control in patients with type 2 diabetes (T2D) who had poor glucose control despite treatment with OHA, patients were divided into two groups: those in whom the use of all OHA except SU was suspended [the SU(+) group; n = 10] and those in whom all OHA, including SU, were discontinued [the SU(−) group; n = 12]. Twenty-two patients with T2D undergoing treatment at Saiseikai Niigata Second Hospital were included in the study. Patients had been treated with maximum doses of OHA, but had failed to show adequate glucose control (HbA1c ≥7%). All patients refused inpatient therapy, but agreed to receive LM-50 injections t.i.d. on an outpatient basis. All patients provided informed consent for inclusion in the present study, which was an open-label, randomized, active-controlled, parallel-group, comparative study. For patients in the SU(+) group, SU was continued at the same dose and LM-50 injections were started at a dose of 0.1 units/kg, t.i.d. Patients in the SU(−) group were started on the same dose of LM-50, but all OHAs were discontinued. The primary efficacy endpoint was the change in HbA1c level. After 1–2 months of LM-50 injections, patients began to get used to the self-monitoring of blood glucose levels and the dose of LM-50 injected was adjusted with the aim of reducing preprandial blood glucose levels to ≤130 mg/dL. Changes in HbA1c levels for patients in both the SU(+) and SU(−) groups are shown in Fig. 1. For the 10 patients in the SU(+) group, significant decreases in HbAlc started to be seen after 1 months treatment with LM-50. Similarly, patients in the SU(−) group exhibited significant decreases after 1 months treatment with LM-50. At the end of 12 months, significant improvements were observed in both the SU(+) and SU(−) groups, with the effects of LM-50 t.i.d. almost the same, regardless of whether SU was continued or discontinued. In the SU(+) group, the percentage of patients in whom HbA1c fell to <7.0% or <6.5% was 50% and 20%, respectively, compared with 66.7% and 41.7%, respectively, in the SU(−) group. There was no significant difference in the percentage change in HbA1c or the percentage of patients in whom HbA1c fell to <7.0% and <6.5% between the two groups. Figure 1 Mean HbA1c levels in patients in whom the use of all oral hypoglycemic agents (OHA), except sulfonylureas (SU), was suspended [SU(+) group; n = 10; ◆] and those in whom all OHA, including SU, were discontinued [SU(−) group; n = 12; ]. ... The body mass index (BMI) and insulin dose administered increased significantly in both the SU(+) and SU(−) groups over the 12-month treatment period (Table 1). There were no significant differences in terms of the percentage change in BMI or the insulin dose between the two groups (Table 1). Table 1 Baseline characteristics and the outcomes and changes from baseline after 12 months treatment of patients in whom the use of all oral hypoglycemic agents (OHA), except sulfonylureas (SU), was suspended [SU(+) group] and those in whom all OHA, ... Furthermore, although the incidence of slight hypoglycemia was the same in the SU(+) and SU(−) groups, serious hypoglycemia did not occur in any of the patients in either group during the 12-month observation period. In general, it is believed that when insulin therapy is introduced on an outpatient basis, complete discontinuation of SU treatment often results in a decrease in the amount of insulin that enters the portal vein, thereby exacerbating hyperglycemia. However, this scenario was not observed in the present study, probably because the LM-50 used in the present study contained both an ultraquick-acting component and an intermediate-acting component (in a ratio of 1:1) and the intermediate-acting component acted in lieu of the basal secretory component induced by SU treatment. Because the LM-50 used in the present study contains bolus insulin and basal insulin at a ratio of 1:1, it is believed that this provides insulin in a pattern that approximates physiological secretion in humans.1,2 In conclusion, LM-50 injections administered three times daily produced almost the same effects in patients with initially poor glycemic control on OAH, regardless of whether SU was continued.

Mucosal Immune Function Comparison Between Amenorrheic and Eumenorrheic Distance Runners

This study examined the effects of amenorrhea on mucosal immune function and susceptibility to upper respiratory tract infection (URTI) in elite female distance runners. Based on their menstrual cycles during the prior year, 21 elite, collegiate, female distance runners were designated as eumenorrheic runners (ERs; n = 8; 19.9 ± 0.8 years) or amenorrheic runners (ARs; n n = 13; 20.0 ± 0.3 years). Resting saliva and blood samples were collected in the morning. The secretory immunoglobulin A (SIgA) concentration was measured using enzyme-linked immunosorbent assay. The SIgA secretion rate was calculated. Serum 17β-estradiol concentrations and serum progesterone concentrations were measured using radioimmunoassay. Subjects reported the appearance of URTI symptoms (sore throat, headache, runny nose, coughing, or fever), if any, during the prior month. The serum estradiol concentration and salivary SIgA secretion rate were significantly lower for ARs than for ERs (p < 0.05). Serum progesterone concentration was not significantly different between groups. Higher frequencies of headache, runny nose, coughing, and fever were observed in ARs than in ERs. Results show that athletic amenorrhea with low estrogen might accelerate downregulation of mucosal immune function in athletes and enhance susceptibility to infection.

GENISTEIN DIET: EFFECTS ON JEJUNUM FROM THE R117H CYSTIC FIBROSIS MICE

We have previously shown wild‐type (Wt) C57BL/6J mice fed a genistein‐containing diet (600 mg/kg food, 600G) for 1‐month, have significantly elevated basal levels of jejunal chloride (Cl) secretion (measured as transepithelial short circuit current, Isc) compared to mice fed a genistein‐free diet (0G); a gender‐specific response to 600G, i.e. females produced a significant increase in Cl secretion, whereas no response was observed in males. We fed male and female cystic fibrosis R117H mutant mice, 600G and 0G diets for 1‐month. Basal Isc was unchanged in 600G male mice (40.4 ± 15.2 μA/cm2, n = 7) compared to 0G controls (34.9 ± 7.7 μA/cm2, n = 9). Basal Isc was significantly increased in 600G female mice (39.1 ± 7.4 μA/cm2, n = 3) compared to 0G controls (24.8 ± 3.0 μA/cm2, n = 3). Comparable steady‐state increases in Isc in response to forskolin (10 μm, bilateral) were observed in 600G and 0G male and female mice. There was no effect of the inhibitor of the Na+/K+/2Cl− co‐transporter, bumetanide (100 μm, basolateral), on the forskolin‐stimulated Isc in either 600G or 0G fed male and female mice. Addition of acetazolamide (100 μm, bilateral) to block the HCO3− secretory component, produced an increase in the percentage inhibition in 600G fed males (11.9 ± 3.0 %, n = 7) and 600G fed females (13.7 ± 1.9 %, n = 3), compared to their respective controls. In the male R117H mice there were no changes in jejunal morphology associated with the diet groups (villi length, number of goblet cells per villi, crypt depth and number of goblet cells per crypt). These data suggest that, in male R117H mice, a 4‐week diet treatment with 600G does not provide any benefits towards the overall magnitude of basal or forskolin‐mediated secretion, but in female R117H mice there is improvement in basal Isc with the 600G diet. Sarah Polito was supported by the Midwestern University Summer DO Fellowship Program. Layla Al‐Nakkash was supported by the Soy Health Research Fund.

A Chinese family carrying novel mutations in SEC23B and HFE2, the genes responsible for congenital dyserythropoietic anaemia II (CDA II) and primary iron overload, respectively

A Chinese family carrying novel mutations in <scp><i>SEC23B</i></scp> and <scp><i>HFE2</i></scp>, the genes responsible for congenital dyserythropoietic anaemia <scp>II</scp> (<scp>CDA II</scp>) and primary iron overload, respectively

Crofelemer for the treatment of secretory diarrhea

Secretory diarrhea is a leading cause of morbidity and mortality worldwide. Crofelemer is a first-in-class antidiarrheal agent that simultaneously targets two distinct channels, the cystic fibrosis conductance regulator and calcium-activated chloride channel, responsible for chloride and fluid secretion in the GI tract. Crofelemer is a novel compound extracted from the stem bark latex of the Croton lechleri tree found in the western Amazonian region of South America. There is little to no systemic absorption of crofelemer when given orally and studies have shown minimal toxicity beyond mild gastrointestinal effects. In studies in diarrheal illness associated with primarily a secretory component, such as cholera, travelers’ diarrhea and acute infectious diarrhea, crofelemer has shown improvements in stool consistency and duration of symptoms. Less clear, but interesting, results have been observed in other diarrheal diseases associated with a mixed pathology, including diarrhea in patients with HIV and diarrhea-predominant irritable bowel syndrome.