Evaluation of T and B lymphocyte function in clinical practice using a flow cytometry based proliferation assay

The golden standard for functional evaluation of immunodeficiencies is the incorporation of [³H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [³H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA + SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.     

The study protocol of: ‘Initiating end of life care in stroke: clinical decision-making around prognosis’

Background

The initiation of end of life care in an acute stroke context should be focused on those patients and families with greatest need. This requires clinicians to synthesise information on prognosis, patterns (trajectories) of dying and patient and family preferences. Within acute stroke, prognostic models are available to identify risks of dying, but variability in dying trajectories makes it difficult for clinicians to know when to commence palliative interventions. This study aims to investigate clinicians’ use of different types of evidence in decisions to initiate end of life care within trajectories typical of the acute stroke population.

Methods/design

This two-phase, mixed methods study comprises investigation of dying trajectories in acute stroke (Phase 1), and the use of clinical scenarios to investigate clinical decision-making in the initiation of palliative care (Phase 2). It will be conducted in four acute stroke services in North Wales and North West England. Patient and public involvement is integral to this research, with service users involved at each stage.

Discussion

This study will be the first to examine whether patterns of dying reported in other diagnostic groups are transferable to acute stroke care. The strengths and limitations of the study will be considered. This research will produce comprehensive understanding of the nature of clinical decision-making around end of life care in an acute stroke context, which in turn will inform the development of interventions to further build staff knowledge, skills and confidence in this challenging aspect of acute stroke care.

     

Human Antibody Responses to the Polyclonal Dryvax Vaccine for Smallpox Prevention Can Be Distinguished from Responses to the Monoclonal Replacement Vaccine ACAM2000

Dryvax (Wyeth Laboratories, Inc., Marietta, PA) is representative of the vaccinia virus preparations that were previously used for preventing smallpox. While Dryvax was highly effective, the national supply stocks were depleted, and there were manufacturing concerns regarding sterility and the clonal heterogeneity of the vaccine. ACAM2000 (Acambis, Inc./Sanofi-Pasteur Biologics Co., Cambridge, MA), a single-plaque-purified vaccinia virus derivative of Dryvax, recently replaced the polyclonal smallpox vaccine for use in the United States. A substantial amount of sequence heterogeneity exists within the polyclonal proteome of Dryvax, including proteins that are missing from ACAM2000. Reasoning that a detailed comparison of antibody responses to the polyclonal and monoclonal vaccines may be useful for identifying unique properties of each antibody response, we utilized a protein microarray comprised of approximately 94% of the vaccinia poxvirus proteome (245 proteins) to measure protein-specific antibody responses of 71 individuals receiving a single vaccination with ACAM2000 or Dryvax. We observed robust antibody responses to 21 poxvirus proteins in vaccinated individuals, including 11 proteins that distinguished Dryvax responses from ACAM2000. Analysis of protein sequences from Dryvax clones revealed amino acid level differences in these 11 antigenic proteins and suggested that sequence variation and clonal heterogeneity may contribute to the observed differences between Dryvax and ACAM2000 antibody responses.     

Appendicitis as the presenting manifestation of Kawasaki disease

In cases of Kawasaki’s disease (KD) presenting as acute surgical abdomen, rarely has the presence of acute appendicitis been found. We report two cases of histologically confirmed acute appendicitis in the presence of KD and a review of the literature as it pertains to acute abdomen and atypical presentations of KD.     

Academic Proficiency in Children After Early Congenital Heart Disease Surgery

Children with early surgery for congenital heart disease (CHD) are known to have impaired neurodevelopment; their performance on school-age achievement tests and their need for special education remains largely unexplored. The study aimed to determine predictors of academic achievement at school age and placement in special education services among early CHD surgery survivors. Children with CHD surgery at <1 year of age from January 1, 1998 to December 31, 2003, at the Arkansas Children’s Hospital were identified. Out-of-state births and infants with known genetic and/or neurologic conditions were excluded. Infants were matched to an Arkansas Department of Education database containing standardized assessments at early school age and special-education codes. Predictors for achieving proficiency in literacy and mathematics and the receipt of special education were determined. Two hundred fifty-six children who attended Arkansas public schools and who had surgery as infants were included; 77.7 % had either school-age achievement-test scores or special-education codes of mental retardation or multiple disabilities. Scores on achievement tests for these children were 7–13 % lower than those of Arkansas students (p < 0.01). They had an eightfold increase in receipt of special education due to multiple disabilities [odds ratio (OR) 10.66, 95 % confidence interval (CI) 4.23–22.35] or mental retardation (OR 4.96, 95 % CI 2.6–8.64). Surgery after the neonatal period was associated with decreased literacy proficiency, and cardiopulmonary bypass during the first surgery was associated with decreased mathematics proficiency. Children who had early CHD surgery were less proficient on standardized school assessments, and many received special education. This is concerning because achievement-test scores at school age are “real-world” predictors of long-term outcomes.     

Roles of notch signalling in mandibular condylar cartilage

Importance

Notch proteins are cell surface transmembrane spanning receptors which mediate critically important cellular functions through direct cell–cell contact. Interactions between Notch receptors and their ligands regulate cell fate decisions such differentiation, proliferation and apoptosis in numerous tissues. We have previously shown using immunohistochemistry that Notch1 is localized primarily to the prechondroblastic (chondroprogenitor) layer of the mandibular condylar cartilage (MCC).

Objective

To test if Notch signalling changes patterns of proliferation and differentiation in the MCC and to investigate if Notch signalling acts downstream of Fibroblast Growth Factor 2 (FGF-2).

Methods

Condylar cartilage explants were cultured over serum-free DMEM containing either 0 or 50 nM DAPT, a Notch signal inhibitor. Explants were used for RNA extraction and immunohistochemistry.

Results

Analysis of gene array data demonstrated that the perichondrial layer of the MCC is rich in Notch receptors (Notch 3 and 4) and Notch ligands (Jagged and Delta) as well as various downstream facilitators of Notch signalling. Disruption of Notch signalling in MCC explants decreased proliferation (Cyclin B1 expression) and increased chondrocyte differentiation (Sox9 expression). Moreover, we found that the actions of FGF-2 in MCC are mediated in part by Notch signalling.

Conclusion

These data suggest that Notch signalling contributes to the regulation of proliferation and differentiation in the MCC.