Efficacy and Safety of Once-Daily Insulin Degludec/Insulin Aspart versus Insulin Glargine (U100) for 52 Weeks in Insulin-Naïve Patients with Type 2 Diabetes: A Randomized Controlled Trial.

PURPOSE: The efficacy and safety of insulin degludec/insulin aspart (IDegAsp) once daily (OD) compared with insulin glargine U100 (IGlar) OD over 52 weeks in insulin-naïve adults with type 2 diabetes mellitus (T2DM) was investigated. METHODS: In this open-label, parallel-group treat-to-target trial, participants were randomized (1:1) to receive IDegAsp OD (breakfast, n = 266) or IGlar OD (as per label, n = 264). Participants then entered a 26-week extension phase (IDegAsp OD, n = 192; IGlar OD, n = 221). The primary endpoint was change from baseline to Week 26 in HbA1c. RESULTS: After 26 and 52 weeks, mean HbA1c decreased to similar levels in both groups. After 52 weeks, the mean estimated treatment difference was -0.08% (-0.26, 0.09 95%CI), confirming the non-inferiority of IDegAsp OD versus IGlar OD evaluated at Week 26. After 52 weeks, there was a similar reduction in mean fasting plasma glucose in both treatment groups. The rate of confirmed hypoglycemic episodes was 86% higher (p < 0.0001) whereas the rate of nocturnal hypoglycemia was 75% lower (p < 0.0001) for IDegAsp versus IGlar. CONCLUSION: Nocturnal-confirmed hypoglycemia was higher with IGlar whereas overall and diurnal hypoglycemia were higher with IDegAsp dosed at breakfast. These results highlight the importance of administration of IDegAsp with the main meal of the day, tailored to the individual patient's needs. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01045707 [core]) and NCT01169766 [ext].

Assessment for ease of use and preference of a new prefilled insulin pen (FlexTouch Degludec U100/U200) versus the SoloSTAR insulin pen by patients with diabetes and healthcare professionals.

OBJECTIVE: FlexTouch® (FT) is a new prefilled insulin pen with no push-button extension and a low injection force used to deliver several basal insulins, including insulin degludec across a wide dose range (1 - 80 units with FT 100 IU/ml [FT100] and 2 - 160 units with 200 IU/ml [FT200]). This study was carried out to evaluate whether the novel features of FT affect the preferences of the device among patients with diabetes and healthcare professionals compared with the widely used SoloSTAR® pen. RESEARCH DESIGN AND METHODS: A multicenter, randomized, open-label, crossover study compared FT100 and FT200 with SoloSTAR. The study included patients with either type 1 (n = 22) or type 2 diabetes (n = 42), nurses (n = 32) and physicians (n = 32). Subjects were randomized to test each of the FT100, FT200 and SoloSTAR pens in a crossover set up. Subjects performed injections into a foam cushion at 4 - 6 different doses per device (2, 20, 40, 80, 120 and 160 IU). RESULTS: Overall, a significantly higher proportion of subjects, including dexterity-impaired and pen-naive patients, preferred to use FT100 (93.0%; 119/128) and FT200 (91.4%; 117/128) compared with 2.3% (3/128) and 3.9% (5/128) who preferred SoloSTAR (p < 0.001), respectively. CONCLUSION: FT100 and FT200 were preferred over SoloSTAR by nurses, physicians and patients with diabetes. This may be due to the novel design of FT, which improves ease of use, preference and confidence in delivering a complete, accurate dose of insulin, even at high doses.

Efficacy and Safety of Insulin Degludec 200 U/mL and Insulin Degludec 100 U/mL in Patients with Type 2 Diabetes (Begin: Compare).

OBJECTIVE: The purpose of the present study was to provide clinical data on the efficacy and safety of insulin degludec (IDeg) 200 U/mL compared with IDeg 100 U/mL in patients with type 2 diabetes mellitus (T2DM) currently treated with basal insulin in combination with oral antidiabetic drugs. METHODS: In this 22-week, treat-to-target trial, eligible adult patients with T2DM were randomized 1:1 to IDeg 200 or IDeg 100 U/mL once daily (OD) (n = 186 and 187, respectively). The starting insulin dose was based on a 1:1 transfer of the total prerandomization basal insulin dose. The primary endpoint was change (%) from baseline in glycosylated hemoglobin A1C (A1C) after 22 weeks of treatment. RESULTS: A total of 373 subjects (mean age 59.8 years, A1C 8.2%, fasting plasma glucose 149.6 mg/dL [8.3 mmol/L], body mass index 33.3 kg/m2) were randomized. A1C reduction with IDeg 200 U/mL was noninferior to that of IDeg 100 U/mL (IDeg 200 U/mL - IDeg 100 U/mL estimated treatment difference: -0.11%, 95% confidence interval (CI): -0.28 to 0.05). Rates of overall confirmed hypoglycemia were low and similar between both formulations (5.17 and 5.66 events/patient-year of exposure [PYE] for IDeg 200 and 100 U/mL, respectively). Similarly, the rates of nocturnal confirmed hypoglycemia were low (1.27 and 1.70 events/PYE for 200 and 100 U/mL). In general, both IDeg formulations were well tolerated (respective rates of adverse events: 4.16 and 3.00 events/PYE for 200 and 100 U/mL). CONCLUSION: The 200 and 100 U/mL formulations of IDeg provide comparable and effective levels of glycemic control with similar, low rates of overall confirmed and nocturnal confirmed hypoglycemia.

Injection force and dose accuracy of FlexTouch for the delivery of a new basal insulin.

OBJECTIVE: The aim of the paper is to determine the dose accuracy and injection force of FlexTouch (FT) filled with insulin degludec 100 U/ml, insulin degludec 200 U/ml and insulin degludec/insulin aspart 100 U/ml, SoloStar (SS) filled with insulin glargine 100 U/ml and KwikPen (KP) filled with insulin lispro mix 75/25 100 U/ml. METHODS: Dose accuracy was measured at minimum, midpoint and maximum doses (FT 1, 2, 40, 80 and 160 U; SS 1, 40 and 80 U; KP 1, 30 and 60 U). Injection force was measured during the injection of the maximum dose. RESULTS: All doses delivered from FT were within ISO limits (ISO 11608-1:2012) for degludec 100 U/ml, degludec 200 U/ml and degludec/aspart 100 U/ml, and the pens delivered insulin accurately and consistently at all doses tested. Similarly, all tested doses from KP filled with insulin lispro mix 75/25 100 U/ml were within ISO limits, while some doses from SS filled with insulin glargine 100 U/ml were outside ISO limits. FT had a significantly lower injection force than SS and KP (p < 0.05). CONCLUSIONS: FT filled with insulin degludec and insulin degludec/insulin aspart, delivered insulin accurately and consistently within ISO limitations at all doses tested; similarly, KP delivered insulin within ISO limitations at all doses tested and SS delivered most doses within ISO limitations. The significantly lower injection force of FT compared to SS and KP is an important feature that has the potential to make the injection process easier for people with diabetes.

Insulin degludec once-daily in type 2 diabetes: simple or step-wise titration (BEGIN: once simple use).

INTRODUCTION: Insulin degludec (IDeg) is a new basal insulin in development with a flat, ultra-long action profile that may permit dosing using a simplified titration algorithm with less frequent self-measured blood glucose (SMBG) measurements and more simplified titration steps than currently available basal insulins. METHODS: This 26-week, multi-center, open-label, randomized, treat-to-target study compared the efficacy and safety of IDeg administered once-daily in combination with metformin in insulin-naïve subjects with type 2 diabetes using two different patient-driven titration algorithms: a "Simple" algorithm, with dose adjustments based on one pre-breakfast SMBG measurement (n = 111) versus a "Step-wise" algorithm, with adjustments based on three consecutive pre-breakfast SMBG values (n = 111). IDeg was administered using the FlexTouch® insulin pen (Novo Nordisk A/S, Bagsværd, Denmark), with once-weekly dose titration in both groups. RESULTS: Glycosylated hemoglobin (HbA1c) decreased from baseline to week 26 in both groups (-1.09%, IDegSimple; -0.93%, IDegStep-wise). IDegSimple was non-inferior to IDegStep-wise in lowering HbA1c [estimated treatment difference (IDegSimple - IDegStep-wise): -0.16% points (-0.39; 0.07)95% CI]. Fasting plasma glucose was reduced (-3.27 mmol/L, IDegSimple; -2.68 mmol/L, IDegStep-wise) with no significant difference between groups. Rates of confirmed hypoglycemia [1.60, IDegSimple; 1.17, IDegStep-wise events/patient year of exposure (PYE)] and nocturnal confirmed hypoglycemia (0.21, IDegSimple; 0.10, IDegStep-wise events/PYE) were low, with no significant differences between groups. Daily insulin dose after 26 weeks was 0.61 U/kg (IDegSimple) and 0.50 U/kg (IDegStep-wise). No significant difference in weight change was seen between groups by week 26 (+1.6 kg, IDegSimple; +1.1 kg, IDegStep-wise), and there were no clinically relevant differences in adverse event profiles. CONCLUSION: IDeg was effective and well tolerated using either the Simple or Step-wise titration algorithm. While selection of an algorithm must be based on individual patient characteristics and goals, the ability to attain good glycemic control using a simplified titration algorithm may enable patient empowerment through self-titration, improved convenience, and reduced costs.

Accuracy and preference assessment of prefilled insulin pen versus vial and syringe with diabetes patients, caregivers, and healthcare professionals.

OBJECTIVES: The primary objective of this study was to investigate the dosing accuracy of the new prefilled FlexTouch insulin pen (FT) in comparison to conventional vial and syringe (V&S) when used by patients (Pts), caregivers (CG) and healthcare professionals (HCPs). METHODS: A total of 120 subjects participated in the trial (40 diabetes patients aged 61 ± 11 [mean ± SD] yrs, 20 caregivers [parents and other relatives], 20 physicians, and 40 nurses/certified diabetes educators). The participants were introduced to the devices in randomized order and were asked to perform injections of 5, 25, 43 and 79 IU doses into laboratory tubes. Dosing accuracy was analyzed by weighing the tubes on a pharmaceutical balance and calculating the mean absolute deviation (MAD) from the intended doses. After completing a device assessment questionnaire, Patient Perception Questionnaire (PPQ), with questions regarding device design and performance, the procedure was repeated for the other device, and the patients were finally asked to complete a device preference questionnaire (DPQ). RESULTS: Dosing accuracy was significantly better for FT when used by any of the cohorts at all doses. (MAD ± SD for FT/V&S; 5 IU: 0.4 ± 0.4/0.6 ± 0.6 IU; 25 IU: 0.3 ± 0.4/0.7 ± 0.9 IU; 43 IU: 0.4 ± 0.4/0.9 ± 1.2 IU; 79 IU: 0.5 ± 0.5/1.7 ± 1.6 IU, p < 0.005 for all doses). Dosing accuracy with FT for all three subgroups was comparable (patients: 0.35-0.59 IU; HCP&CG: 0.29-0.54 IU; n.s.). Dosing accuracy with V&S for all three subgroups was not comparable: HCP and CG performed much better with V&S than patients and delivered the doses with significantly higher accuracy (range of mean MAD; patients: 0.81-2.54 IU; HCP&CG: 0.51-1.30 IU, p < 0.005 at all doses). FT was ranked superior to V&S for all aspects of the PPQ. In the DPQ, 93% of the patients voted for FT (neutral: 5%, V&S: 2%), (CG: 100%/0%/0%; HCPs: 85%/2%/13%; p < 0.001 in all cases). CONCLUSION: FT, compared to V&S, was more accurate at all tested doses and was used with similar accuracy by patients, HCPs, and CGs. Using questionnaires only, and without dexterity assessment, study participants rated FT higher than V&S in every component of the PPQ and the vast majority of them preferred FT. These findings may point to a better alternative for dosing accuracy and improved adherence when using the new prefilled insulin pen compared to V&S for insulin delivery in patients with diabetes.

Empowering people with diabetes: improving perceptions and outcomes with technical advances in insulin pens.

For people with diabetes treated with insulin, the development of insulin pens has led to important advantages compared with the use of vials and syringes. Insulin pens are associated with improved ease of use, user confidence, treatment satisfaction, and quality of life compared with vials and syringes. Continual improvements to insulin pen designs to further enhance usability and improve patient perceptions may help to lower patients' resistance to initiating insulin therapy and further improve treatment adherence. This article reviews recent developments in prefilled insulin pens that may assist health care professionals when considering insulin-delivery devices to recommend to their patients.

Comparison of patient preference for two insulin injection pen devices in relation to patient dexterity skills.

BACKGROUND: Impaired dexterity has been reported to be prevalent in diabetes patients independent from the existence of diabetic neuropathy. This study was performed to investigate the impact of dexterity impairment on patient preference for two insulin pen injection devices (InnoLet and FlexTouch). METHODS: Ninety patients [54 male/36 female; age (mean ± standard deviation), 62 ± 8 years; disease duration, 18 ± 11 years; hemoglobin A1c, 7.2 ± 1.0%] were included in this investigation and were stratified into four different groups based on the results of a dexterity test (Jebsen-Taylor Hand Function Test) and assessment of visual impairment: 15 type 1 (group A) and 30 type 2 (group B) patients with impaired dexterity, 30 type 1/type 2 patients with visual impairment (group C), and 15 type 1/type 2 patients without any impairment (group D). The patients performed a cognitive function test (number connection test), were introduced to the devices in random order, and were asked to perform some mock injections before completing a six-item standardized preference questionnaire. RESULTS: There was a strong preference for FlexTouch in all groups. All unimpaired patients (100%, group D) preferred FlexTouch, as did the vast majority in all other groups. Only 11% of the patients with impaired cognitive function preferred InnoLet, as did a few patients with more severely impaired dexterity or with visual impairment (group A, 13%; group B, 3%; group C, 14%). CONCLUSIONS: Patient dexterity skills may have an influence on device preference, especially if the impairment is more pronounced.

Patient device assessment evaluation of two insulin injection devices in a mixed cohort of insulin-treated patients with type 1 or type 2 diabetes mellitus.

OBJECTIVE: FT (FlexTouch*) is a new disposable insulin injection pen device for use in insulin-treated patients with diabetes mellitus. The aim of this study was to evaluate patient perception of FT versus IL (InnoLet†) with respect to the ease of use and patient preference in a mixed patient cohort with different kinds and degrees of visual or dexterity impairments. METHODS: Ninety patients were included into this investigation (54 male/36 female, age [mean ± SD]: 62 ± 8 yrs, disease duration: 18 ± 11 yrs, HbA1c: 7.2 ± 1.0%). After assessment of visual acuity and dexterity skills (by Jebsen-Taylor Hand Function Test), the patients were introduced to the two pen devices in random order, and were asked to perform mock injections with 10 IU, 30 IU and 50 IU doses before completing a 41 item standardized device assessment questionnaire. The questions asked were covering five topics of pen use (confidence in delivering a correct dose, dose setting, performance of the injection, general handling, and others) and could be answered with a rank scale from '1 = very easy' to '5 = very difficult'. RESULTS: FT was ranked superior to IL with respect to the injection procedure (FT: 1.2 ± 0.1 vs. IL: 2.1 ± 0.4, p < 0.001) and general handling (1.3 ± 0.2 vs. 2.3 ± 0.7, p < 0.001), and numerically better with respect to confidence in correct dosing (1.4 ± 0.2 vs. 2.1 ± 0.9, n.s.). The two devices were ranked equally for ease of dose setting (1.6 ± 0.3 vs. 1.7 ± 0.4, n.s.). When ranked individually, FT use was recommended by 92.2% of the patients (IL: 30.0%). KEY LIMITATIONS: Patients of this investigation were from one local area (San Jose, CA, USA) only. The subgroups may be considered small for the performed analysis. CONCLUSIONS: In summary, FT was perceived to be easier to use than IL in this investigation.

Healthcare professional and patient assessment of a new prefilled insulin pen versus two widely available prefilled insulin pens for ease of use, teaching and learning.

OBJECTIVE: FlexTouch * (FT) is a new prefilled insulin pen with no push-button extension at any set dose and a low activation force that is designed to improve ease of use and insulin administration. This paper reports the results of two usability studies assessing perceptions of FT compared with KwikPen † (KP)and SoloStar ‡ (SS) among healthcare professionals (HCPs; both physicians and nurses) and people with diabetes (both insulin pen-experienced and insulin pen-naïve). RESEARCH DESIGN AND METHODS: Participants were randomly assigned to start with FT or KP in one study and FT or SS in the other. Participants performed injections at different doses (20, 40 and 60 International Units [IU] in the FT vs. KP study or 20, 40 and 80 IU in the FT vs. SS study) into a foam cushion before answering questions on ease of use, teaching and learning, confidence and preference. RESULTS: A total of 59 people with diabetes and 61 HCPs took part in the FT vs. SS study, and 79 people with diabetes and 81 HCPs took part in the FT vs. KP study. Considerably more patients and HCPs rated FT as very/fairly easy to inject with than KP or SS, particularly at the maximum dose (≥80% vs. ≤38% and ≤23%, respectively), and more were very/rather confident in the ability to manage daily insulin injections with FT than KP or SS. Overall, FT was rated significantly higher for ease of teaching and learning to use than KP or SS (all p < 0.001 vs. FT), and was preferred for teaching and learning compared with KP or SS (≥39% vs. ≤4% and ≤6%, respectively). More patients and HCPs would recommend FT (≥95%) than KP (≤72%) or SS (≤71%). The same pattern was generally seen across physicians, nurses, insulin pen-experienced and pen-naïve participants. CONCLUSIONS: The findings suggest that devices such as FT are easy to use and can be prescribed with relatively few training needs, which may improve ease of insulin initiation, increase pen use, and ultimately improve treatment adherence. A limitation of the usability questionnaire used in this study is that it did not assess the factors that influence preference. Further analyses could be conducted to determine the factors that appeal to different users.

FlexTouch: a prefilled insulin pen with a novel injection mechanism with consistent high accuracy at low- (1 U), medium- (40 U), and high- (80 U) dose settings.

BACKGROUND: FlexTouch® is the only prefilled insulin pen that utilizes an easy touch button that does not extend at any dose in place of a push-button extension. Rigorous testing has shown that the new FlexTouch pen accurately and consistently delivers insulin doses. METHODS: This study assessed dose accuracy of FlexTouch, KwikPen®, and SoloSTAR®. Dose accuracy for minimum, medium, and maximum doses of each pen type (1, 40, and 80 U for FlexTouch and SoloSTAR and 1, 30, and 60 U for KwikPen) was assessed. RESULTS: FlexTouch delivered all doses consistently, as demonstrated by low standard deviations. FlexTouch showed similar accuracy to KwikPen at 1 U and to SoloSTAR at 40 and 80 U. However, FlexTouch was significantly more accurate at delivering 1 U of insulin than SoloSTAR (p < .0001). CONCLUSIONS: This study demonstrates that FlexTouch, a new prefilled pen, delivers insulin accurately and consistently at low, medium, and high doses. In addition, FlexTouch is currently the only prefilled pen that has a push button that does not extend at any dose, making FlexTouch easier to use than other pens.

Ease of use and preference of a new versus widely available prefilled insulin pen assessed by people with diabetes, physicians and nurses.

OBJECTIVE: FlexTouch® (FT; Novo Nordisk A/S, Bagsvaerd, Denmark) is a new prefilled insulin pen that has no push-button extension and low injection force. This multi-centre, crossover usability study evaluated the perceptions of, and preference for, FT versus another widely used prefilled pen, SoloStar® (SS; Sanofi, Paris, France), by people with diabetes and healthcare professionals. RESEARCH DESIGN AND METHODS: Following instruction, participants performed injections into a foam cushion, randomly alternating between doses of 20, 40 and 80 international units (IU). Participants then answered questions on usability and preference. RESULTS: In all, 59 people with diabetes and 61 healthcare professionals (30 physicians and 31 nurses) took part. Overall, significantly more respondents preferred to use FT than SS (83 vs 10%, respectively), found FT easier to use (83 vs 9%) and would recommend FT to others (83 vs 8%; p < 0.001 for all). More respondents found it 'very/fairly easy' to reach the push-button and to inject 20, 40 and 80 IU with FT (93, 90 and 88% to inject, respectively) than with SS (73, 43 and 15% to inject, respectively; p < 0.001 for all). Most respondents chose FT as giving them the most confidence in correct and complete insulin delivery (76 vs 6%; p < 0.001) and considerably more were 'very/rather confident' in managing their daily insulin injections with FT than with SS (88 vs 58%). CONCLUSIONS: Most participants rated FT as easier to use and to inject with, were more confident in its accuracy of insulin delivery and preferred it to SS.

An assessment of dose accuracy and injection force of a novel prefilled insulin pen: comparison with a widely used prefilled insulin pen.

OBJECTIVE: FlexTouch® (FT) (Novo Nordisk A/S, Bagsværd, Denmark) is a new prefilled insulin pen with a novel injection mechanism encompassing no push-button extension at any dose-setting. This study assessed the dose accuracy and injection force of FT compared with the established Next Generation FlexPen® (NGFP) (Novo Nordisk A/S Bagsværd, Denmark). RESEARCH DESIGN AND METHODS: Dose accuracy was measured at the minimum, medium and maximum doses (FT, 1, 40 and 80 international units (IU) and NGFP, 1, 30 and 60 IU). Injection force was measured during the injection of the maximum dose (FT, 80 IU; NGFP, 60 IU). MAIN OUTCOMES: FT and NGFP delivered insulin accurately and consistently at all doses (mean ± s.d., FT at 1 IU, 0.98 ± 0.07; 40 IU, 39.86 ± 0.33; 80 IU, 79.76 ± 0.64; NGFP at 1 IU, 1.02 ± 0.08; 30 IU, 29.69 ± 0.30; 60 IU, 59.50 ± 0.51). FT had a significantly (p < 0.0001) lower injection force than NGFP. CONCLUSIONS: The study demonstrated that FT and NGFP deliver insulin accurately and consistently at low, medium and high doses. The novel torque spring injection mechanism of FT results in a significantly lower injection force than NGFP and a pen requiring less thumb-pressure to inject insulin may be welcomed by many people with diabetes.

A prefilled insulin pen with a novel injection mechanism and a lower injection force than other prefilled insulin pens.

BACKGROUND: FlexTouch(®) (Novo Nordisk A/S, Bagsvaerd, Denmark) is a new prefilled insulin pen for people with diabetes, with a novel injection mechanism and no push-button extension at any dose setting. This study compared the injection force of FlexTouch with that of SoloStar(®) (sanofi-aventis, Paris, France) and KwikPen(®) (Eli Lilly & Co., Indianapolis, IN). METHODS: Injection force was measured with the manufacturers' recommended needle attached to each pen (NovoFine(®) [Novo Nordisk] 32-gauge tip extra thin wall 6 mm needle for FlexTouch and BD [Franklin Lakes, NJ] MicroFine™ 31-gauge 5 mm needle for SoloStar and KwikPen) during injection of the maximum dose (60 IU for KwikPen and 80 IU for FlexTouch and SoloStar). Injection was performed at three different constant push-button speeds. RESULTS: FlexTouch had a significantly (P<0.0001) lower injection force than SoloStar and KwikPen at all injection speeds. The mean±SD injection force of FlexTouch was 5.1±0.5 N. At 4, 6, and 8 mm/s push-button speeds, the injection force of SoloStar was 13.5±2.1, 19.1±1.9, and 26.9±2.4 N, respectively, and the injection force of KwikPen was 14.5±1.9, 20.9±1.4, and 28.2±1.4 N, respectively. CONCLUSIONS: The injection mechanism of FlexTouch means that insulin injection is driven by a torque spring and not the thumb pressure of the user. This results in a 62-82% lower injection force with FlexTouch than other prefilled insulin pens.

Natural killer T-cell characterization through gene expression profiling: an account of versatility bridging T helper type 1 (Th1), Th2 and Th17 immune responses.

Natural killer T (NKT) cells constitute a distinct lymphocyte lineage at the interface between innate and adaptive immunity, yet their role in the immune response remains elusive. Whilst NKT cells share features with other conventional T lymphocytes, they are unique in their rapid, concomitant production of T helper type 1 (Th1) and Th2 cytokines upon T-cell receptor (TCR) ligation. In order to characterize the gene expression of NKT cells, we performed comparative microarray analyses of murine resting NKT cells, natural killer (NK) cells and naïve conventional CD4+ T helper (Th) and regulatory T cells (Treg). We then compared the gene expression profiles of resting and alpha-galactosylceramide (alphaGalCer)-activated NKT cells to elucidate the gene expression signature upon activation. We describe here profound differences in gene expression among the various cell types and the identification of a unique NKT cell gene expression profile. In addition to known NKT cell-specific markers, many genes were expressed in NKT cells that had not been attributed to this population before. NKT cells share features not only with Th1 and Th2 cells but also with Th17 cells. Our data provide new insights into the functional competence of NKT cells which will facilitate a better understanding of their versatile role during immune responses.

Effect of B7-2 and CD40 signals from activated antigen-presenting cells on the ability of zwitterionic polysaccharides to induce T-Cell stimulation.

Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria elicit potent CD4+-T-cell responses in vivo and in vitro. We demonstrated that HLA-DR on professional antigen-presenting cells (APCs) is required for ZPS-induced T-cell proliferation in vitro (15). Recently, it was shown that ZPSs are processed to low-molecular-weight carbohydrates by a nitric oxide-mediated mechanism in endosomes and locate in the major histocompatibility complex class II pathway (5, 15). The effect of the ZPS-mediated expression of HLA-DR and costimulatory molecules on the APC and T-cell engagement and subsequent T-cell activation has not been elucidated. Herein, we report that ZPS-mediated induction of HLA-DR-surface expression and T-cell proliferation are maximally enhanced after incubation of APCs for 8 h with ZPS. Treatment of APCs with bafilomycin A inhibits the up-regulation of ZPS-mediated HLA-DR surface expression and leads to inhibition of T-cell proliferation. Monoclonal antibodies (MAbs) to the costimulatory molecules B7-2 and CD40L specifically block ZPS-mediated T-cell activation, while a MAb to B7-1 does not. Surface expression of B7-2 and B7-1 but not of CD40 is maximally enhanced at 8 to 16 h of treatment of APCs with ZPS. The results demonstrate that the cellular immune response to ZPS depends on the translocation of HLA-DR to the cell surface and requires costimulation via B7-2 and CD40 on activated APCs. The implication is that activation of ZPS-specific T cells requires an orchestrated arrangement of both presenting and costimulatory molecules to form an immunological synapse.

Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1d-restricted T cells.

A group of T cells recognizes glycolipids presented by molecules of the CD1 family. The CD1d-restricted natural killer T cells (NKT cells) are primarily considered to be self-reactive. By employing CD1d-binding and T cell assays, the following structural parameters for presentation by CD1d were defined for a number of mycobacterial and mammalian lipids: two acyl chains facilitated binding, and a polar head group was essential for T cell recognition. Of the mycobacterial lipids tested, only a phosphatidylinositol mannoside (PIM) fulfilled the requirements for CD1d binding and NKT cell stimulation. This PIM activated human and murine NKT cells via CD1d, thereby triggering antigen-specific IFN-gamma production and cell-mediated cytotoxicity, and PIM-loaded CD1d tetramers identified a subpopulation of murine and human NKT cells. This phospholipid, therefore, represents a mycobacterial antigen recognized by T cells in the context of CD1d.

Zwitterionic polysaccharides stimulate T cells by MHC class II-dependent interactions.

Polysaccharides of pathogenic extracellular bacteria commonly have negatively charged groups or no charged groups at all. These molecules have been considered classic T cell-independent Ags that do not elicit cell-mediated immune responses in mice. However, bacterial polysaccharides with a zwitterionic charge motif (ZPSs), such as the capsular polysaccharides of many strains of Bacteroides fragilis, Staphylococcus aureus, and Streptococcus pneumoniae type 1 elicit potent CD4(+) T cell responses in vivo and in vitro. The cell-mediated response to ZPS depends on the presence of both positively charged and negatively charged groups on each repeating unit of the polysaccharide. In this study, we define some of the requirements for the presentation of ZPS to CD4(+) T cells. We provide evidence that direct interactions of T cells with APCs are essential for T cell activation by ZPS. Monocytes, dendritic cells, and B cells are all able to serve as APCs for ZPS-mediated T cell activation. APCs lacking MHC class II molecules do not support this activity. Furthermore, mAb to HLA-DR specifically blocks ZPS-mediated T cell activation, while mAbs to other MHC class II and class I molecules do not. Immunoprecipitation of lysates of MHC class II-expressing cells following incubation with ZPS shows binding of ZPS and HLA-DR. Electron microscopy reveals colocalization of ZPS with HLA-DR on the cell surface and in compartments of the endocytic pathway. These results indicate that MHC class II molecules expressing HLA-DR on professional APCs are required for ZPS-induced T cell activation. The implication is that binding of ZPS to HLA-DR may be required for T cell activation.