The 2.0-Å resolution crystal structure of a trimeric antibody fragment with noncognate VH–VL domain pairs shows a rearrangement of VH CDR3

The 2.0-Å resolution x-ray crystal structure of a novel trimeric antibody fragment, a “triabody,” has been determined. The trimer is made up of polypeptides constructed in a manner identical to that previously described for some “diabodies”: a V_L domain directly fused to the C terminus of a V_H domain—i.e., without any linker sequence. The trimer has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. For the particular structure reported here, the polypeptide was constructed with a V_H domain from one antibody fused to the V_L domain from an unrelated antibody giving rise to “combinatorial” Fvs upon formation of the trimer. The structure shows that the exchange of the V_L domain from antibody B1-8, a V_λ domain, with the V_L domain from antibody NQ11, a V_κ domain, leads to a dramatic conformational change in the V_H CDR3 loop of antibody B1-8. The magnitude of this change is similar to the largest of the conformational changes observed in antibody fragments in response to antigen binding. Combinatorial pairing of V_H and V_L domains constitutes a major component of antibody diversity. Conformationally flexible antigen-binding sites capable of adapting to the specific CDR3 loop context created upon V_H–V_L pairing may be employed by the immune system to maximize the structural diversity of the immune response.     

Causes of Death and Risk Factors for Mortality in Patients With Severe Chronic Obstructive Pulmonary Disease

ObjectiveThe objective of this study was to assess the causes of death and risk factors for mortality in a cohort of patients with severe chronic obstructive pulmonary disease (COPD).Patients and methodsWe studied 203 patients with severe COPD (forced expiratory volume in 1 second [FEV₁] <50%), who were attended in our respiratory department day hospital (2001-2006). Clinical variables were recorded on inclusion, and clinical course and causes of death were retrospectively reviewed.ResultsThe mean (SD) age of patients was 69 (8) years and the mean FEV₁ was 30.8% (8.2%). One-hundred and nine patients died (53.7%); death was attributed to respiratory causes in 72 (80.9%), with COPD exacerbation being the most frequent specific cause within this category (48.3%). During follow-up, 18.7% required admission to the intensive care unit (ICU). Survival at 1, 3, and 5 years was 80%, 53%, and 26%, respectively. The multivariate analysis showed that mortality was associated with age, stage IV classification according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), cor pulmonale, and hospital admission during the year prior to inclusion. Need for admission to the ICU during follow-up was a factor independently associated with higher mortality.ConclusionsMortality in patients with severe COPD was high and exacerbation of the disease was one of the most frequent causes of death. Age, GOLD stage, cor pulmonale, prior admission to hospital, and need for admission to the ICU during follow-up were independent predictors of mortality.     

Influence of tumours on normal cells and optimal chemotherapy regimens: the case of several drugs and toxicity constraints.

Cancer chemotherapy with the application of several drugs is studied. The negative and inhibiting effect of the tumour on normal cells is taken into account. Under certain hypotheses, we determine the optimal regimen that minimizes the tumour burden at the end of a fixed period of therapy while maintaining several normal cell populations above prescribed levels. More precisely, it is demonstrated that the optimal drug administration corresponds to the strategy of intensive chemotherapy.     

Transforming growth factor-β1 and interleukin-10 in breast milk and development of atopic diseases in infants

BACKGROUND: Precise relationship between breastfeeding and infant allergy is poorly understood. Objective Aim was to quantify TGF-beta(1) and IL-10 in colostrum and mature milk from allergic and non-allergic mothers and to verify relationship with allergic disease development. METHODS: Mothers (13 allergics, nine controls) of 22 newborns participated to prospective study on development of children atopy. Colostrum and mature milk were assayed for TGF-beta(1) and IL-10 by ELISA. Children underwent paediatrician evaluation at 6 months of life. RESULTS: Data are presented as median values and range. A significant difference in concentration of TGF-beta(1) between colostrum (330, range 0-3400 pg/mL) and mature milk (215, range 0-2400 pg/mL) was observed in samples from allergic mothers (P=0.015). In mature milk TGF-beta(1) was significantly lower in allergic (215, range 0-2400 pg/mL) than in non-allergic mothers (1059, range 0-6250 pg/mL) (P=0.015). IL-10 was weakly expressed without significant differences between allergic (4.8, range 0-42 and 9.5, range 0-42 pg/mL in colostrum and in mature milk) and non-allergic mothers (0, range 0-42 pg/mL in colostrum and 0, range 0-42 pg/mL in mature milk). After 6 months 46% infants from allergic mothers, but none from controls, presented atopic dermatitis. CONCLUSION: TGF-beta(1) was significantly less secreted in mature milk of allergic mothers, while no difference in IL-10 was found. Particular cytokine patterns in milk could influence development of atopic diseases. Further immunological studies in this field are necessary.     

Hepatoprotective action of prostaglandin A2 analogs under CCl4-induced liver injury in vitro

Aim: The cytoprotective effects of six novel synthetic prostaglandin A(2) analogs against carbon tetrachloride (CCl(4)) as a toxic agent were studied with isolated rat liver hepatocytes in vitro. Results: It was found that hepatocytes treatment with CCl(4) induced: (i) a significant increase of lactic dehydrogenase (LDH) release from cytoplasm; (ii) leakage of glutamate dehydrogenase (GDH) and acid phosphatase from mitochondria and lysosomes, respectively; (iii) 10-fold increase of trien conjugates formation; and (iv) a reduction of free SH-groups by 50%. Prostanoids U-26, U-9 and U-34 decreased cytotoxic index of CCl(4) on average by 1.5-2.0 times and were more effective than PGI(2), the well-known hepatoprotector of prostanoids type. The protective action of the prostanoids was not a cAMP- or Ca(2+)-dependent process. However, prostanoids U-26, U-9 and U-34 normalized intracellular content of SH-groups, reduced trien conjugates formation by 60-80% and strongly prevented enzyme leakage through cellular membranes. They were also able to inhibit CCl(4) effects via decreasing cytochrome P(450)2E1 activity. Conclusion: The results obtained demonstrate that prostanoids provide cytoprotective effects on liver hepatocytes through the prevention of lipid peroxidation of the plasma and the cellular membranes and maintenance of their barrier function.     

Deficiency of P-selectin in a patient with grey platelet syndrome

Abstract: Patient B.G. is a 29-yr-old female with a lifelong bleeding disorder characterized clinically by a highly increased bleeding time, menorrhagias, long-lasting bleeding after cuts and tooth extractions and large post-traumatic haematomas. Her coagulation tests were within normal range, platelet count was 140,000–160,000 per μl, but platelet function was impaired as demonstrated by the absence of collagen-induced aggregation, although no abnormalities were detected in aggregation response to ADP and ristocetin. Morphologically her platelets were characterized by gigantic size – average profile area was about 2.5 times higher than that of control donors, and severe deficiency of α-granules – only 16% of their number in control donors. These features taken together indicated the diagnosis of grey platelet syndrome. As has been shown by quantitative immunoblotting, patient's platelets contained small amounts of α-granule membrane protein P-selectin – about 15% of that in control donors. The content of plasma membrane glycoproteins IIb–IIIa and Ib was not reduced, suggesting the specific deficiency of α-granule membrane protein. Thus, B.G. is the second patient described in the literature (see also Lages et al, J Clin Invest 1991: 87: 919–929) with combined deficiency of α-granules and P-selectin.     

Increased in vivo frequency of IA-2 peptide-reactive IFNgamma+/IL-4- T cells in type 1 diabetic subjects

Active T cell recognition of islet antigens has been postulated as the pathogenic mechanism in human type 1 diabetes, but evidence is scarce. If T cells are engaged, they are expected to display increased clonal size and exhibit a T helper (Th)1/Th2 differentiation state. We used a peptide library that covers tyrosine phosphatase IA-2, a target antigen expressed in pancreatic beta cells, to probe 8 diabetic patients and 5 HLA-matched controls. When tested in a high resolution IFNgamma/IL-4 double color ELISPOT assay directly ex vivo, the number of IA-2-reactive IFNgamma producing cells was 17-fold higher in patients than in controls and IL-4 producing cells were not present. An average of 9 peptides was recognized in the patients vs. one in the controls. Determinant recognition primarily involved CD4+ cells and showed high variability among the patients. Furthermore, anti-CD28 antibody signal enhances quantitative assessment of effector T cells in T1D patients. In vitro expansion with peptides and IL-2 results in detection of responding cells in the controls and loss of disease specificity of the T cell response. Together these data provide strong evidence for the active targeting of IA-2 by Th1 memory effector cells in human type 1 diabetes.