Interleukin-12 (IL-12) is a cytokine that promotes cell-mediated immunity to intracellular pathogens by inducing type 1 helper T cell (TH1) responses and interferon-gamma (IFN-gamma) production. IL-12 binds to high-affinity beta1/beta2 heterodimeric IL-12 receptor (IL-12R) complexes on T cell and natural killer cells. Three unrelated individuals with severe, idiopathic mycobacterial and Salmonella infections were found to lack IL-12Rbeta1 chain expression. Their cells were deficient in IL-12R signaling and IFN-gamma production, and their remaining T cell responses were independent of endogenous IL-12. IL-12Rbeta1 sequence analysis revealed genetic mutations that resulted in premature stop codons in the extracellular domain. The lack of IL-12Rbeta1 expression results in a human immunodeficiency and shows the essential role of IL-12 in resistance to infections due to intracellular bacteria.

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91 phox ) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91 phox gene revealed a single-base mutation (C 3 T) at position ؊53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position ؊53 of the gp91 phox promoter, and the mutation at position ؊53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position ؊50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position ؊56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions ؊53 and ؊50 significantly reduced the gp91 phox promoter activity; however, the mutation at position ؊56 did not affect the promoter activity. In transient cotransfection study, PU

In extensive preclinical testing, a CD3 x CD19 bispecific antibody (BsAb) induced killing of malignant B cells by resting T cells even in an autologous situation. In a 14 day clonogenic assay using a CD19+ pre-B cell line (REH), BsAb required repeated administration together with IL-2 to achieve a 5 log kill by resting peripheral blood T cells. Intravenously administered BsAb in an intrapatient dose escalation study of 3 patients with B cell non-Hodgkin's lymphoma showed limited toxicity (WHO grade II fever and chills) due to tumor necrosis factor-alpha (TNF-alpha) release by T cells. Pharmacokinetics with 2.5 mg BsAb showed peak levels of 200-300 micrograms/ml and a t1/2 of 10.5 h. The next patient, with chronic lymphocytic leukemia (CLL), received 0.6 mg BsAb/m2 as an i.v. infusion preceded by 1 MU IL-2/m2 s.c. Improved T cell activation was noted, as indicated by an increase in IFN-gamma, IL-6, IL-8, and IL-10, in addition to high TNF-alpha increases. TNF-alpha increases were highest on the first day. Toxicity remained restricted to grade II fever and chills, observed every day after the infusion of BsAb. No clear clinical effects were seen in this chemotherapy-resistant CLL patient with a high tumor burden. If subsequent patients also show limited toxicity, treatment of patients with a lower tumor load seems to be warranted to evaluate the efficacy of CD3 x CD19 BsAb therapy.

Background External quality assessment (EQA) programs for general chemistry tests have evolved from between laboratory comparison programs to trueness verification surveys. In the Netherlands, the implementation of such programs has reduced inter-laboratory variation for electrolytes, substrates and enzymes. This allows for national and metrological traceable reference intervals, but these are still lacking. We have initiated a national endeavor named NUMBER (Nederlandse UniforMe Beslisgrenzen En Referentie-intervallen) to set up a sustainable system for the determination of standardized reference intervals in the Netherlands. Methods We used an evidence-based 'big-data' approach to deduce reference intervals using millions of test results from patients visiting general practitioners from clinical laboratory databases. We selected 21 medical tests which are either traceable to SI or have Joint Committee for Traceability in Laboratory Medicine (JCTLM)-listed reference materials and/or reference methods. Per laboratory, per test, outliers were excluded, data were transformed to a normal distribution (if necessary), and means and standard deviations (SDs) were calculated. Then, average means and SDs per test were calculated to generate pooled (mean±2 SD) reference intervals. Results were discussed in expert meetings. Results Sixteen carefully selected clinical laboratories across the country provided anonymous test results (n=7,574,327). During three expert meetings, participants found consensus about calculated reference intervals for 18 tests and necessary partitioning in subcategories, based on sex, age, matrix and/or method. For two tests further evaluation of the reference interval and the study population were considered necessary. For glucose, the working group advised to adopt the clinical decision limit. Conclusions Using a 'big-data' approach we were able to determine traceable reference intervals for 18 general chemistry tests. Nationwide implementation of these established reference intervals has the potential to improve unequivocal interpretation of test results, thereby reducing patient harm.

BackgroundThe prognostic value of biochemical tests in critically ill patients with multiple organ failure and suspected bowel ischemia is unknown.MethodsIn a prospective observational cohort study intensive care patients were included when the attending intensivist considered intestinal ischemia in the diagnostic workup at any time during intensive care stay. Patients were only included once. When enrolment was ended each patient was classified as ‘proven intestinal ischemia’, ‘ischemia likely’, ‘ischemia unlikely’ or ‘no intestinal ischemia’. Proven intestinal ischemia was defined as the gross disturbance of blood flow in the bowel, regardless of extent and grade. Classification was based on reports from the operating surgeon, pathology department, endoscopy reports and CT-scan. Lactate dehydrogenase (LDH), creatine kinase (CK), alanine aminotransferase (ALAT), L-lactate were available for the attending physician. D-lactate and intestinal fatty acid binding protein (I-FABP) were analysed later in a batch. I-FABP was only measured in patients with proven ischemia or no ischemia.ResultsFor 44 of the 120 included patients definite diagnostic studies were available. 23/44 patients (52%) had proven intestinal ischemia as confirmed by surgery, colonoscopy, autopsy and/or histopathological findings. LDH in these patients was 285 U/l (217–785) vs 287 U/l (189–836) in no-ischemia; p = 0.72. CK was 226 U/l in patients with proven ischemia (126–2145) vs 347 U/l (50–1427), p = 0.88. ALAT was 53 U/l (18–300) vs 34 U/l (14–34), p-0,56. D-lactate 0.41 mmol/l (0.11-0.75) vs 0.56 mmol/l (0.27-0.77), p = 0.46. L-lactate 3.5 mmol/l (2.2-8.4) vs 2.6 mmol/l (1.7-3.9), p = 0.09. I-FABP 2872 pg/ml (229–4340) vs 1020 pg/ml (239–5324), p = 0.98. Patient groups proven and likely ischemia together compared to unlikely and no-ischemia together showed significant higher L-lactate (p = 0.001) and higher D-lactate (p = 0.003).ConclusionsMeasurement of LDH, CK, and ALAT did not discriminate critically ill patients with proven intestinal ischemia from those with definite diagnosis no-ischemia. However, L-lactate and D-lactate levels were higher in patients with proven or likely ischemia and need further study just as I-FABP.

Background: Accurate measurements of rheumatoid factors (RFs), autoantibodies binding IgG, are important for diagnosing rheumatoid arthritis (RA) and for predicting disease course. Worldwide, various RF assays are being used that differ in technique and target antigens. We studied whether assay choice leads to clinically important discrepancies in RF status and level. Methods: RF measurements using four commercial RF assays were compared in 32 RF+ samples. Using enzyme-linked immunosorbent assays (ELISAs), the influence of the target antigen source – human IgG (hIgG) versus rabbit IgG (rIgG) – on measured RF levels was investigated in arthralgia patients and RA patients. Results: Substantial discrepancies were found between RF levels measured in the four commercial assays. Six samples (19%) with RF levels below or slightly above the cutoff in the rIgG-based Phadia assay were RF+ in three assays using hIgG as the target antigen, some with very high levels. Direct ELISA comparisons of RF reactivity against hIgG and rIgG estimated that among 173 ACPA+ arthralgia patients, originally RF negative in rIgG-based assays, up to 10% were single positive against hIgG. Monoclonal RFs binding to hIgG and rIgG or hIgG only supported these findings. In a cohort of 69 early RA patients, virtually all RF responses reacted with both targets, although levels were still variable. Conclusions: The use of RF assays that differ in technique and target antigen, together with the different specificities of RF responses, leads to discrepancies in RF status and levels. This has important consequences for patient care if RA diagnosis and disease progression assessments are based on RF test results.

A bispecific antibody directed to T and B cells (CD3 x CD19 bsAb) was daily infused intravenously in escalating doses from 10 micrograms up to 5 mg in three patients with chemotherapy-resistant non-Hodgkin lymphoma; in this way we aimed to activate T cells to kill the malignant B cells. Only limited toxicity was observed, consisting of moderate fever preceded by chills or shivers and mild thrombocytopenia. No human anti-(mouse Ig) antibodies were found. Pharmacokinetics showed a t1/2 of 10.5 h with peak levels of 200-300 ng/ml after infusion of 2.5 mg bsAb. bsAb in serum was functionally active in vitro. After bsAb infusion a rise in serum tumour necrosis factor alpha was observed, accompanied by an increase in soluble CD8 and to some extent in soluble interleukin-2 receptor (IL-2R), but not in interferon gamma. IL-4 or soluble CD4. No evidence was found for monocyte activation (no increases in IL-6, IL-8 or IL-1 beta in serum). No gross changes in histology or number of IL-2R+, CD4+ or CD8+ cells were found in the lymph nodes after therapy, but one patient showed activated CD8+ T cells within the tumour nodules. In conclusion, after intravenously administered CD3 x CD19 bsAb only moderate toxicity was found, probably due to CD8+ T cell activation and cytokine release, without CD4+ T cell activation.

We previously reported that a CD3 x CD19 bispecific antibody (bsAb) can induce efficient killing of tumour cells by preactivated T cells isolated from patients with B cell malignancy. For future intravenous application we investigated whether resting T cells from peripheral blood can be stimulated to proliferate and become cytotoxic with the CD3 x CD19 bsAb alone. Indeed peripheral blood mononuclear cells, isolated from healthy donors or patients with B cell malignancy, started to proliferate within 1 day in response to CD3 x CD19 bsAb. Within the same time span cytotoxic activity against CD19-positive tumour cells was already detectable. Maintenance of cytotoxic activity was seen during 3 days of culture but optimal lysis of the target cells then required fresh CD3 x CD19 bsAb in the cytotoxicity assay. Essentially the same results for proliferation and cytotoxicity were found when separated CD4-positive and CD8-positive T cells were activated by the bsAb in the presence of autologous monocytes. These results may be relevant for the in vivo application of the bsAb when used as immunotherapy in patients with B cell malignancy.