The Effect of Renal Impairment on the Pharmacokinetics and Pharmacodynamics of Ertugliflozin in Subjects With Type 2 Diabetes Mellitus.

Ertugliflozin is a highly selective and potent inhibitor of the sodium-glucose cotransporter 2 in development for the treatment of type 2 diabetes mellitus. The glycemic efficacy of sodium-glucose cotransporter 2 inhibitors such as ertugliflozin depends on glucose filtration through the kidney. This phase 1, open-label study evaluated the effect of renal impairment on the pharmacokinetics, pharmacodynamics, and tolerability of ertugliflozin (15 mg) in type 2 diabetes mellitus and healthy subjects with normal renal function (estimated glomerular filtration rate not normalized for body surface area ≥90 mL/min) and type 2 diabetes mellitus subjects with mild (60-89 mL/min), moderate (30-59 mL/min), or severe (<30 mL/min) renal impairment (n = 36). Blood and urine samples were collected predose and over 96 hours postdose for pharmacokinetic evaluation and measurement of urinary glucose excretion over 24 hours. Log-linear regression analyses indicated predicted mean area under the concentration-time curve values for mild, moderate, and severe renal function groups that were ≤70% higher relative to subjects with normal renal function. Generally consistent results were obtained with categorical analysis based on analysis of variance. The increase in ertugliflozin exposure in subjects with renal impairment is not expected to be clinically meaningful. Regression analysis of change from baseline in urinary glucose excretion over 24 hours vs estimated glomerular filtration rate showed a decrease in urinary glucose excretion with declining renal function. A single 15-mg dose of ertugliflozin was well tolerated in all groups.

Bioequivalence of fixed-dose combination RIN®-150 to each reference drug in loose combination.

BACKGROUND: RIN(®)-150 is a fixed-dose combination (FDC) tablet containing rifampicin (RMP, 150 mg) and isoniazid (INH, 75 mg) developed for the treatment of tuberculosis. SETTING: This study was conducted at a single center: the Pfizer Clinical Research Unit in Singapore. OBJECTIVE: To demonstrate bioequivalence of each drug component between RIN-150 and individual products in a loose combination. DESIGN: This was a randomized, open-label, single-dose, two-way crossover study. Subjects received single doses of RIN-150 or two individual reference products under fasting conditions in a crossover fashion, with at least 7 days washout between doses. The primary measures for comparison were peak plasma concentration (Cmax) and the area under plasma concentration-time curve (AUC). RESULTS: Of 28 subjects enrolled, 26 completed the study. The adjusted geometric mean ratios of Cmax and AUClast between the FDC and single-drug references and 90% confidence intervals were respectively 91.63% (90%CI 83.13-101.01) and 95.45% (90%CI 92.07-98.94) for RMP, and 107.58% (90%CI 96.07-120.47) and 103.45% (90%CI 99.33-107.75) for INH. Both formulations were generally well tolerated in this study. CONCLUSION: The RIN-150 FDC tablet formulation is bioequivalent to the two single-drug references for RMP and INH at equivalent doses.

Bioequivalence of fixed-dose combination Myrin®-P Forte and reference drugs in loose combination.

BACKGROUND: Myrin®-P Forte is a fixed-dose combination (FDC) tablet containing rifampicin (RMP, 150 mg), isoniazid (INH, 75 mg), ethambutol (EMB) hydrochloride (275 mg) and pyrazinamide (PZA, 400 mg) developed for the treatment of tuberculosis (TB). SETTING: This study was conducted at a single centre--the Pfizer Clinical Research Unit in Singapore. OBJECTIVE: To demonstrate the bioequivalence of each drug component of the Myrin-P Forte FDC and the individual product in loose combination. DESIGN: In a randomized, open-label, single-dose, two-way, crossover study, subjects received single doses of Myrin-P Forte or four individual products under fasting conditions in a crossover fashion with at least 7 days washout between doses. The primary measures for comparison were peak plasma concentration (C(max)) and the area under plasma concentration-time curve (AUC). RESULTS: Of 36 subjects enrolled, 35 completed the study. The adjusted geometric mean ratios and 90% confidence intervals for C(max) and AUC values were completely contained within bioequivalence limits (80%, 125%) for all four drugs in both formulations. Both treatments were generally well tolerated in the study. CONCLUSION: The Myrin-P Forte FDC tablet formulation is bioequivalent to the four single-drug references for RMP, INH, EMB hydrochloride and PZA at equivalent doses.

Familial aggregation of food allergy and sensitization to food allergens: a family-based study.

BACKGROUND: The increasing prevalence of food allergy (FA) is a growing clinical and public health problem. The contribution of genetic factors to FA remains largely unknown. OBJECTIVE: This study examined the pattern of familial aggregation and the degree to which genetic factors contribute to FA and sensitization to food allergens. METHODS: This study included 581 nuclear families (2,004 subjects) as part of an ongoing FA study in Chicago, IL, USA. FA was defined by a set of criteria including timing, clinical symptoms obtained via standardized questionnaire interview and corroborative specific IgE cut-offs for > or =95% positive predictive value (PPV) for food allergens measured by Phadia ImmunoCAP. Familial aggregation of FA as well as sensitization to food allergens was examined using generalized estimating equation (GEE) models, with adjustment for important covariates including age, gender, ethnicity and birth order. Heritability was estimated for food-specific IgE measurements. RESULTS: FA in the index child was a significant and independent predictor of FA in other siblings (OR=2.6, 95% CI: 1.2-5.6, P=0.01). There were significant and positive associations among family members (father-offspring, mother-offspring, index-other siblings) for total IgE and specific IgE to all the nine major food allergens tested in this sample (sesame, peanut, wheat, milk, egg white, soy, walnut, shrimp and cod fish). The estimated heritability of food-specific IgE ranged from 0.15 to 0.35 and was statistically significant for all the nine tested food allergens. CONCLUSION: This family-based study demonstrates strong familial aggregation of FA and sensitization to food allergens, especially, among siblings. The heritability estimates indicate that food-specific IgE is likely influenced by both genetic and environmental factors. Together, this study provides strong evidence that both host genetic susceptibility and environmental factors determine the complex trait of IgE-mediated FA.

Tight regulation from a single tet-off rAAV vector as demonstrated by flow cytometry and quantitative, real-time PCR.

Vectors suitable for delivery of therapeutic genes to the CNS for chronic neurodegenerative diseases will require regulatable transgene expression. In this study, three self-regulating rAAV vectors encoding humanized green fluorescent protein (hGFP) were made using the tetracycline (tet)-off system. Elements were cloned in different orientations relative to each other and to the AAV internal terminal repeat (ITRs). The advantage of this vector system is that all infected cells will carry both the 'therapeutic' gene and the tet-regulator. To compare the efficiency of the vectors, 293T cells infected by each vector were grown in the presence or absence of the tet-analog doxycycline (dox). Cells were analyzed by flow cytometry for hGFP protein expression, and quantitative RT-PCR (QRT-PCR) for levels of hGFP mRNA and the tet-activator (tTA) mRNA. In the presence of dox, cells infected with one of the vectors, rAAVS3, showed less than 2% total fluorescent intensity and mRNA copy number than cells grown without dox. The other two vectors were significantly more leaky. Levels of tTA mRNA were not affected by dox. The S3 vector also displayed tight regulation in HeLa and HT1080 cells. To assess regulation in the brain, the S3 vector was injected into rat striatum and rats maintained on regular or dox-supplemented water. At 1 month after vector injection, numerous positive cells were observed in rats maintained on regular water whereas only rare positive cells with very low levels of fluorescence were observed in rats maintained on water containing dox. The QRT-PCR analysis showed that dox inhibited expression of hGFP mRNA in brain by greater than 99%. These results demonstrate that exceedingly tight regulation of transgene expression is possible using the tet-off system in the context of a self-regulating rAAV vector and that the specific orientation of two promoters relative to each other and to the ITRs is important. Regulatable vectors based on this design are ideal for therapeutic gene delivery to the CNS.

Rapid immune reconstitution following autologous hematopoietic stem cell transplantation in children: a single institution experience.

In this retrospective study, we review the immune reconstitution of children undergoing autologous hematopoietic stem cell transplantation. A total of 125 patients underwent autologous transplantation between 1992 and 2000. The report includes data on 58 patients. Data were not available on the remaining patients who either died before testing or data were not obtained. The parameters evaluated include: (a) immunophenotype by flow cytometry to quantify lymphocyte subpopulations (b) mitogen stimulation assays, and (c) quantitative immunoglobulins. The analysis reveals that CD3+ cells did not reach the normal range during the first year post-transplant. The median percentage of CD4+ cells was below normal up to 6 months post-transplant, while the absolute number remain low throughout the first year. The CD8+ percentage and absolute numbers remain normal at all times post-transplant. The CD19+ cells were also normal post-transplantation. The mitogen lymphocyte stimulation was normal in 27 out of 31 patients tested after 6 months post-transplant. Our analysis of immune reconstitution shows a similar pattern to previous studies with a faster recovery of the CD4/CD8 ratio, especially in those patients who did not receive TBI. In conclusion, the observed deficiencies are transient and have very little clinical significance because, historically, the rate of serious infections is low despite prolonged immune suppression. The recovery post-autologous transplant is fast.

Abnormal CD40 ligand (CD154) expression in human immunodeficiency virus-infected children.

The CD40 ligand (CD154), expressed primarily on activated CD4-positive T cells, is a costimulatory molecule involved in B-cell proliferation, germinal center formation, and immunoglobulin class switching. Since B-cell abnormalities including hypergammaglobulinemia and abnormal antibody-specific immune responses are prominent and occur early during the course of pediatric human immunodeficiency virus (HIV) infection, we measured the baseline levels and the induced levels of expression of CD154 on CD3(+) CD8(-) (T helper cells) in HIV-infected children and uninfected children born to HIV-positive mothers. The percentage of CD154(+) T helper cells activated in vitro and the level of CD154 expressed per T helper cell (mean fluorescent channel [MFC]) were significantly lower in the HIV-infected children than in the uninfected control group (77% +/- 3% versus 89% +/- 1%, respectively [P < 0.002], and 261 +/- 174 versus 415 +/- 130 MFC, respectively [P < 0.03]). The levels of CD154 expressed on resting T helper cells in the HIV-infected group were not significantly different from the levels observed in the control group. In the HIV-infected children, the level of CD154 on activated T helper cells correlated with the level of immunodeficiency, as assessed by the CD4 T-cell levels (correlation coefficient [r] = 0.707, P = 0.003), but did not correlate with the viral load or with any of the serum immunoglobulin concentrations measured in this group of HIV-infected children. The baseline level of CD154 expressed on T helper cells did, however, correlate with the concentration of immunoglobulin A in serum. We conclude that HIV-infected children have impaired regulation of CD154 expression which may contribute to the immune dysregulation commonly observed.

Surface and cytoplasmic immunoglobulin expression in circulating B-lymphocytes in acute Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis of young childhood predominantly affecting the coronary arteries. IgA plasma cells have been found to infiltrate vascular and nonvascular tissues in fatal acute KD. To determine whether IgA B-lymphocytes were increased in the peripheral blood of patients with KD, we performed three-color flow cytometry to detect surface and cytoplasmic immunoglobulin expression (IgA, IgM, IgD, and IgG) of peripheral B-lymphocytes in KD patients during the acute, subacute, and convalescent stages of illness and in age-matched febrile and afebrile pediatric controls. Surprisingly, absolute numbers of B-lymphocytes expressing IgA were found to be significantly lower in peripheral blood of acute KD patients compared with febrile and afebrile pediatric controls. These findings indicate that IgA plasma cells are not present in KD tissue as a result of excess numbers of these IgA B-lymphocytes in peripheral blood. We speculate that IgA B-lymphocytes are selectively withdrawn from the peripheral circulation into KD target tissues as part of a specific IgA immune response.

Epidermal growth factor receptor glycosylation is required for ganglioside GM3 binding and GM3-mediated suppression [correction of suppresion] of activation.

Gangliosides are able to bind to the epidermal growth factor receptor and inhibit its activation, but the mechanism of this inhibition is unknown. To address the role of receptor carbohydrates in facilitating interaction with gangliosides, we examined the ability of GM3 to bind the deglycosylated receptor and inhibit its autophosphorylation. Flow cytometry studies demonstrated that deglycosylation of the receptor did not affect its ability to be transported to the cell membrane. In contrast with the native (fully glycosylated) receptor, GM3 did not coimmunoprecipitate with the deglycosylated receptor. Using a novel colorimetric bead binding assay, GM3 was shown to bind well to the immunoprecipitated native receptor but not at all to the deglycosylated receptor. Finally, the addition of GM3 to cells with deglycosylated epidermal growth factor receptors did not result in significant further inhibition of autophosphorylation of the receptor, despite a 10-fold decrease in phosphorylation of the native epidermal growth factor receptor by 200 microM GM3. These studies suggest that ganglioside affects epidermal growth factor receptor activity through a direct interaction that requires receptor glycosylation, and contribute to our understanding of the role of gangliosides in cell membrane function.

Coexpression of normal and mutated CD40 ligand with deletion of a putative RNA lariat branchpoint sequence in X-linked hyper-IgM syndrome.

We describe a novel CD40 ligand (CD40L) splicing mutation in a patient with X-linked hyper-IgM syndrome (X-HIM) associated with alternate splicing of exon 3, resulting in the expression of both full-length and exon-3-skipped CD40L mRNA. The mutation is an 8-bp deletion 25 bp upstream of the intron 2/exon 3 junction which overlaps a putative RNA branchpoint, suggesting that it may impair RNA lariat formation. The exon-3-skipped CD40L transcript encodes a truncated protein (CD40LDeltaE3) encompassing the cytoplasmic, transmembrane, and extracellular stalk domains, but lacking the CD40L receptor binding domain. CD40LDeltaE3 protein expression was readily detectable in transfected Cos cells by immunofluorescence. In cells cotransfected with CD40LDeltaE3 and wild-type CD40L, expression of CD40LDeltaE3 did not inhibit the expression of wild-type CD40L monomers, but strongly inhibited staining by the conformationally sensitive anti-CD40L mAb 5c8, suggesting that CD40LDeltaE3 acts in a dominant negative manner to inhibit the assembly of functional CD40L trimers. This mechanism may contribute to the pathophysiology of CD40L deficiency in X-HIM patients with leaky splice site mutations.

Apoptosis occurs in isolated and banked primary mouse hepatocytes.

Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.

Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women.

The placenta may play a critical role in inhibiting vertical transmission of HIV-1. Here we demonstrate that leukemia inhibitory factor (LIF) is a potent endogenous HIV-1-suppressive factor produced locally in placentae. In vitro, LIF exerted a potent, gp130-LIFRbeta-dependent, HIV coreceptor-independent inhibition of HIV-1 replication with IC50 values between 0.1 pg/ml and 0.7 pg/ml, depending on the HIV-1 isolate. LIF also inhibited HIV-1 in placenta and thymus tissues grown in ex vivo organ culture. The level of LIF mRNA and the incidence of LIF protein-expressing cells were significantly greater in placentae from HIV-1-infected women who did not transmit HIV-1 to their fetuses compared with women who transmitted the infection, but they were not significantly different from placentae of uninfected mothers. These findings demonstrate a novel pathway for endogenous HIV suppression that may prove to be an effective immune therapy for HIV infection.

Measurement of CD40 ligand (CD154) expression on resting and in vitro-activated T cells.

Measurement of the ability of in vitro activated T lymphocytes to express CD40 ligand (CD154) is a valuable tool for diagnosis of the X-linked hyper-IgM syndrome (XHIM) and for investigating abnormalities in the costimulatory functions of T lymphocytes. Resting peripheral blood lymphocytes do not normally express any appreciable levels of CD154, necessitating in vitro activation. This unit details the in vitro activation procedure, the three-color monoclonal antibody panels used to label the appropriate lymphocyte subsets, the controls required to interpret the assay, the setup and acquisition of listmode data files, and the gating and analysis protocols used to interpret the data.

Clinically relevant functional flow cytometry assays.

As a lymphocyte proceeds along the pathway of cell division from the very earliest events of receptor aggregation-kinase activation to the final physical process of cell division, several physiologic functions occur, which can be measured by flow cytometry. Many of the functions along this pathway can be induced and measured in vitro and have led to the development of clinically relevant tests, which have been reviewed as functional flow cytometry procedures. It must be noted, however, that in addition to all of the physiologic processes that occur in lymphocytes (and in fact most cells) along the pathway to proliferation there are also several differentiated immune functions that are subject to flow cytometric analysis. Procedures for the evaluation of immune functions, such as phagocytosis, cellular aggregation, natural killer-cell cytotoxicity, cytokine secretion, antibody secretion, and antigen-specific cellular cytotoxicity assays have all been described.

Postexercise immune correlates in children with and without cystic fibrosis.

INTRODUCTION: Previous studies have shown that children with cystic fibrosis (CF) are capable of mounting a normal immune response after the stress of exercise. However, few data are available regarding the underlying mechanisms by which this immune modulation occurs. METHODS: In this study, lymphocyte and leukocyte cell counts were measured before and immediately after a single bout of exhaustive exercise in 25 children (ages 8-17 yr; 12 with CF and 13 healthy controls). Catecholamine, cortisol, and insulin levels, age, nutritional parameters, and static and dynamic lung function were measured as potential correlates for immune modulation. We hypothesized that catecholamine levels would be associated with the immune changes seen after exercise in children with CF. RESULTS: Our results demonstrated positive correlations between age and the change in cell counts after exercise for white blood cells (r = 0.44, P < 0.03), lymphocytes (r = 0.60, P < 0.002), monocytes (r = 0.43, P < 0.03), and CD3-CD16+CD56+ cells (r = 0.61, P < 0.002). Lower increases in the lymphocyte and CD3-CD16+CD56+ cells were observed in the CF group. Changes in pre- and post-exercise norepinephrine levels were weakly correlated with the changes in granulocyte, lymphocyte, and monocyte cell counts. Changes in cortisol levels correlated with lymphocyte and CD19+ cell count changes for the CF group but not for the healthy controls. Within the CF group, the severity of lung disease (as indicated by a FEV1) was negatively correlated with changes in lymphocyte (r = -0.66, P < 0.02) and CD3-CD16+CD56+ cell counts (r = -0.67, P < 0.02). CONCLUSION: The results suggest that postexercise changes in cell counts occur in an age dependent, norepinephrine associated manner. Disease severity for children with CF also appears to enhance the postexercise leukocytosis with pronounced increases seen in natural killer cells.

Decreased levels of CD54 (ICAM-1)-positive lymphocytes in the peripheral blood in untreated patients with active juvenile dermatomyositis.

Significant abnormalities are observed in the peripheral blood of juvenile dermatomyositis (JDM) patients with active disease. In this study, we confirm that there is a significant increase in the relative percentage of B lymphocytes in the peripheral blood of a group of untreated children with newly diagnosed active JDM compared to healthy children (P < 0.0001). In order to investigate if properties intrinsic to B cells contributed to their relative increase in JDM, the percentage of B cells expressing activation markers (CD23, CD25, CD54, and CD69) was measured and compared to pediatric controls. Compared to healthy children less than 10 years of age (not significantly different from the JDM group), the JDM patients had an increase in the proportion of lymphocytes expressing CD19 (B cells; P = 0.0017) and decreases in the percentage of lymphocytes that were CD3(-) CD16(+) and/or CD56(+) (NK cells; P = 0. 01) and CD3(+) CD8(+) (T suppressor/cytotoxic cells; P = 0.02). There were no significant differences in any of the B-cell activation markers assessed. Of note, the percentage of CD54(+) non-B lymphocytes (i.e., T cells and NK cells expressing CD54) was significantly lower in the JDM patients (25% +/- 5%) than in the "age-related" healthy control group (43% +/- 4%; P = 0.013). These results suggest the following for untreated children with active JDM: (i) the increase in the percentage of peripheral blood B cells is not due to intrinsic B-cell activation, and (ii) CD54/ICAM-1(+) non-B cells, CD8(+) T cells, and NK cells are being removed from circulation and may be participating in the pathophysiology of the disease.

Immune modulation following aerobic exercise in children with cystic fibrosis.

Previous studies have demonstrated altered immune response following exercise in healthy adults and children. As data are lacking in children with cystic fibrosis, we evaluated the immune response following acute exercise and hypothesized that acute increases in cellular changes would be seen but would be blunted in subjects with CF. Leukocytes, lymphocytes, and their subsets as well as natural killer cell number and activity were determined before, immediately after, and one hour post exhaustive exercise in 15 children with cystic fibrosis (8-21 yrs, FEV1 69.5+/-18.0%, colonized with P aeruginosa) and 15 healthy controls (8-18 yrs, FEV1 107.5+/-10.7%). At baseline the cystic fibrosis group had greater leukocytes (9.25+/-2.83 vs. 5.17+/-0.96 x 10(9) cells/liter). Immediately post exercise, the cystic fibrosis group demonstrated increases in cell counts for leukocytes (32.4%), lymphocytes (61.8%), granulocytes (36.4%), monocytes (76.2%), and natural killer cells (315%). Similar percentage increases were seen in cell counts for the controls (leukocytes: 39.5%, lymphocytes: 78.5%, granulocytes: 32.0%, monocytes: 75.9%, and NK cells: 442%). Natural killer cell activity also increased by 57.9% in the group with cystic fibrosis and by 43.6% in the healthy controls. Except for elevated leukocyte and granulocyte counts, values returned to baseline at one hour post-exercise. In conclusion, the cellular immune response to acute exercise in children with mild to moderate cystic fibrosis appears normal.