A novel ELISPOT assay to enhance detection of antigen-specific T cells employing antigen-presenting cells expressing vector-driven human B7-1

The ELISPOT assay is used to detect T-cell responses in patients enrolled in vaccine clinical trials; it is a relatively sensitive assay that can be performed without in vitro stimulation (IVS) of PBMC. However, this assay may be limited in some cases because of a weak signal 1 (as is the case for tumor-associated antigens [TAA]), or by a limited number of PBMC available from patients. The objective of this study is to enhance the sensitivity of the ELISPOT by increasing the level of signal 2, in this case, B7-1 expression on antigen-presenting cells (APC), allowing for a more sensitive detection of antigen-specific T-cell precursors. C1RA2 cells were used as APC and were uninfected, or infected with either recombinant avipox (fowlpox)-B7-1 (rF-B7-1) or control fowlpox wild-type (FP-WT) vector. The expressions of B7-1, MHC Class II, as well as HLA-A2 on the infected cells were analyzed by flow cytometry. A nearly threefold increase in B7-1 mean fluorescence intensity (MFI) occurred in the rF-B7-1-infected C1RA2 cells with no changes in MHC Class II or HLA-A2 expression. Various PBMC/APC ratios were used to further analyze the use of rF-B7-1-infected C1RA2 cells as APC in the ELISPOT assay. Fewer APC were required to activate PBMC when C1RA2 infected with rF-B7-1 were used as APC. Furthermore, using a PBMC/APC ratio of 1:1, similar detection was achieved using fewer PBMC. In addition, we demonstrated that the reactivity can be blocked by adding anti-B7-1 antibody. We performed the assay using APC on five normal healthy donors. All five donors showed substantial increases in PF to the Flu matrix peptide (Flu peptide) using the rF-B7-1-infected C1RA2 cells. Finally, we evaluated five cancer patients who received a carcinoembryonic antigen (CEA) vaccine-based therapy. Increases in CEA peptide precursors were noted in all five patients using the B7-1-infected APC. In conclusion, we have demonstrated the ability to enhance the sensitivity of the ELISPOT assay through infection of C1RA2 with rF-B7-1.     

Clinical outcomes of pregnancy in women with type 1 diabetes(1)

OBJECTIVE:To evaluate predictors of neonatal hypoglycemia and macrosomia in 107 consecutive pregnancies in type 1 diabetic women. METHODS:We conducted a case record analysis of singleton type 1 diabetic pregnancies between January 1994 and January 1999 following institution of standardized management. RESULTS:The duration of diabetes in the women was 12.9 +/- 6.8 years, and 44 were primigravidas. The mean HbA1c throughout pregnancy was 7.2 +/- 0.8%. There was no relationship between neonatal blood glucose (checked before the second feed) and HbA1c at any point in pregnancy or mean pregnancy HbA1c (R = 0.20, P >.1). However, there was a negative correlation between neonatal blood glucose and maternal blood glucose during labor (R = -0.33, P <.001). When maternal blood glucose during labor was greater than 8 mM (144 mg/dL), neonatal blood glucose was usually less than 2.5 mM (mean 1.7 +/- 0.4 mM or 31 mg/dL). There was no relationship between mean HbA1c and birth weight (R = 0.02, P >.1) or between maximum insulin dose and birth weight (R = 0.09, P >.1). Fetal abdominal circumference measured by ultrasound at 34 weeks correlated strongly with birth weight (R = 0.72, P <.001). CONCLUSION:Neonatal hypoglycemia correlates with maternal hyperglycemia in labor, not with HbA1c during pregnancy. Macrosomia does not correlate with HbA1c during pregnancy.     

Normal perinatal mortality in type 1 diabetes mellitus in a series of 300 consecutive pregnancy outcomes

Reports of outcomes of pregnancy in women with type 1 diabetes have consistently found increased perinatal mortality and morbidity. The primary objective of our study was to compare the perinatal mortality rate in type 1 diabetic pregnancies with that of the general population. The secondary objective was to compare the morbidities in these groups. A series of 247 women with type 1 diabetes had 300 consecutive pregnancy outcomes analyzed over a 10-year period. They were compared with the control population from the same hospital. Perinatal mortality was 6.6/1000 (95% CI, 0-17), which was significantly lower than the control population rate of 31/1000. There was an increased incidence of morbidity including maternal hypertension, cesarean section, preterm delivery, birth injury, large for gestational age infants, admissions to neonatal intensive care, neonatal hypoglycemia, and phototherapy. Pregnancies in type 1 diabetes can be associated with a normal perinatal mortality rate although morbidity remains elevated compared with controls.     

Ehrlichia chaffeensis seroprevalence among children in the southeast and south-central regions of the United States

BACKGROUND: The reported annual incidence of human monocytic ehrlichiosis, which is due to infection with Ehrlichia chaffeensis, is as high as 5.5 per million in some states, but serosurveys suggest much higher infection rates in some populations. OBJECTIVE: To estimate the prevalence of E chaffeensis infection among children aged 1 to 17 years living in the southeast and south-central United States. DESIGN: Cross-sectional serosurvey. SETTING: Seven academic pediatric medical centers in the southeastern and south-central United States. PATIENTS: Nineteen hundred ninety-nine children (approximately 300 at each center) having their blood drawn for any reason. MAIN OUTCOME MEASURE: The presence of antibody at 2 different cutoff titers to E chaffeensis, as detected by indirect immunofluorescence assay. RESULTS: Overall, 250 children (13%) had E chaffeensis antibody titers of 1:80 or higher and 61 (3%) had titers of 1:160 or higher. Age-adjusted seroprevalence rates varied widely between sites. At 1:80 or higher, the highest rate was in Winston-Salem, NC (22%), and the lowest was in Louisville, Ky (2%). At 1:160 or higher, the highest rate was in Kansas City, Mo (9%), and the lowest was in Oklahoma City, Okla (<1%). In univariate analyses, no associations were found between seroprevalence at either cutoff value and sex, race, source of specimen, or residence demographics. However, age was a significant predictor of seroprevalence at both cutoff values. In multiple logistic regression analysis, study site and age remained strong predictors of seroprevalence, but living in a nonurban ZIP code was not significantly related. CONCLUSION: Infection with E chaffeensis, or related ehrlichiae, may be more common in children than previously recognized.     

Allogeneic bone marrow transplant improves outcome for juvenile myelomonocytic leukaemia

OBJECTIVE: To correlate clinical presentation and therapeutic outcomes in children with a diagnosis of juvenile myelomonocytic leukaemia. METHODS: The medical records of 14 children who fulfilled the International Juvenile Myelomonocytic Leukaemia Working Group Criteria for a diagnosis of juvenile myelomonocytic leukaemia (JMML) presenting to a single institution were reviewed, and their clinical status at September 2000 was documented. RESULTS: The most common presenting features were hepatosplenomegaly and lymphadenopathy. Fifty per cent of cases presented in the first year of life. Nine of 14 patients initially received chemotherapy otherwise used in the treatment of acute myeloid or lymphoblastic leukaemia with no apparent benefit. All six patients who received conditioning therapy with chemotherapy alone, followed by allogeneic bone marrow transplant (BMT), are in complete remission at a median follow-up duration of 12 months (range 5-91 months). Five of six patients surviving post-allogeneic BMT received marrow from an unrelated donor. Only one of seven patients who did not receive BMT survived long-term. CONCLUSION: Children with a diagnosis of JMML should be treated with allogeneic BMT as soon as a suitable donor is found. The role of anti-leukaemic therapy in this disease, prior to BMT, requires further investigation in the context of a multicentre clinical trial.     

Effect of early coronal flaring on working length change in curved canals using rotary nickel-titanium versus stainless steel instruments

This in vitro investigation examined pre- and postinstrumentation working length (WL) measurements in curved root canals. The conditions compared were combinations of (a) stainless steel hand files + Gates Glidden drills (SS) versus nickel-titanium rotary files (Ni-Ti); and (b) early coronal flaring (flaring completed before WL determination) versus late coronal flaring (flaring completed after WL determination). Coronal flaring was accomplished for the SS group using Gates Glidden drills and for the Ni-Ti group using rotary Ni-Ti files (n = 15/group). WL was determined before coronal flaring, immediately after coronal flaring, and again after canal preparation. Results indicated that WL decreased for all canals as a result of canal preparation. The mean decrease in WL was significantly greater for the SS group (-0.48 mm +/- 0.32) than for the Ni-Ti group (-0.22 mm +/- 0.26). Less change in WL occurred in both groups when initial WL was determined after coronal flaring (SS: -0.12 mm +/- 0.13, Ni-Ti: -0.14 mm +/- 0.25).     

The identification and characterization of the marine natural product scytonemin as a novel antiproliferative pharmacophore

Marine natural products provide a rich source of chemical diversity that can be used to design and develop new, potentially useful therapeutic agents. We report here that scytonemin, a pigment isolated from cyanobacteria, is the first described small molecule inhibitor of human polo-like kinase, a serine/threonine kinase that plays an integral role in regulating the G(2)/M transition in the cell cycle. Scytonemin inhibited polo-like kinase 1 activity in a concentration-dependent manner with an IC(50) of 2 microM against the recombinant enzyme. Biochemical analysis showed that scytonemin reduced GST-polo-like kinase 1 activity in a time-independent fashion, suggesting reversibility, and with a mixed-competition mechanism with respect to ATP. Although scytonemin was less potent against protein kinase A and Tie2, a tyrosine kinase, it did inhibit other cell cycle-regulatory kinases like Myt1, checkpoint kinase 1, cyclin-dependent kinase 1/cyclin B, and protein kinase Cbeta2 with IC(50) values similar to that seen for polo-like kinase 1. Consistent with these effects, scytonemin effectively attenuated, without chemical toxicity, the growth factor- or mitogen-induced proliferation of three cell types commonly implicated in inflammatory hyperproliferation. Similarly, scytonemin (up to 10 microM) was not cytotoxic to nonproliferating endotoxin-stimulated human monocytes. In addition, Jurkat T cells treated with scytonemin were induced to undergo apoptosis in a non-cell cycle-dependent manner consistent with its activities on multiple kinases. Here we propose that scytonemin's dimeric structure, unique among natural products, may be a valuable template for the development of more potent and selective kinase inhibitors used for the treatment of hyperproliferative disorders.     

Gene dosage and linezolid resistance in Enterococcus faecium and Enterococcus faecalis

Resistance to linezolid has been associated with a G2576U mutation in domain V of the 23S rRNA. We analyzed nine clinical isolates of linezolid-resistant enterococci and showed a clear association between the number of 23S rRNA genes containing this mutation and the level of linezolid resistance expressed.     

Asperger's syndrome: implications for nursing practice

The most common disorders within the category of Pervasive Developmental Disorders (PDD) are the Autistic Disorders. In the 1994 Diagnostic and Statistical Manual-IV, Asperger's Disorder became a distinct diagnosis within the Autistic category, as part of the disorders first diagnosed in childhood and adolescence. The terms Asperger's Disorder and Asperger's Syndrome are used interchangeably. Attention to and knowledge of Asperger's Syndrome (AS) are necessary to differentiate it from closely related disorders that have overlapping symptoms. There is much discussion about different definitions of Asperger's, different viewpoints of experts in the field, and the diagnostic dilemmas related thereto. Last, and most important, are the nursing implications. Nurses are in a position to identify children with Asperger's early. After identification, the necessary referrals, treatment options, support, and follow-up are essential for these children. Nurses need more knowledge about this disorder and need to be proactive in defining their role to help children with the disorder in the schools and the community.