Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation

The classical complement activation cascade of the immune system is initiated by multivalent binding of its first component, C1q, to the Fc region of immunoglobulins in immune complexes. The C1q binding site on mouse IgG2b has been shown to contain the amino acids Glu 318, Lys 320 and Lys 322 in the C(H)2 domain (Duncan, A.R., Winter, G.,1988. The binding site for C1q on IgG. Nature 322 738-740). Identical or closely related motifs are found on all IgGs in all species, and the binding site has therefore been thought to be universal. However, the results from another study indicate that the site is different in human IgG1 molecules (Morgan, A., Jones, N.D., Nesbitt, A.M., et al., 1995. The N-terminal end of the C(H)2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding. Immunology 86 319-324). To determine the site(s) responsible for complement activation in anti-NIP-mouse/human IgG3 antibodies, we have mutated amino acids Lys 276, Tyr 278, Asp 280, Glu 318, Lys 320 and Lys 322 in two beta-strands in the C(H)2 domains of human IgG3. In addition, we mutated the Glu 333, which resides in close proximity to the postulated C1q-binding site of mouse IgG2b, as well as Leu 235 in the lower hinge region. All mutants were tested in Antibody Dependent Complement Mediated Lysis (ADCML)(4) assays, where the antigen concentration on target cells was varied and human serum was complement source. Only the mutants that lacked the positively charged side chain of lysine in position 322 showed strong reduction in ADCML, particularly at low antigen density on target cells. Alanine scanning of positions 318 and 320 did not affect ADCML, contrary to what was observed for mouse IgG2b. Neither did a leucine to glutamic acid mutation in position 235 have the effect that has been reported for human IgG1. These results suggest that the complement binding site on human IgG3 molecules is different from that found on mouse IgG2b, and possibly on human IgG1 as well. Thus the contact site may not be conserved.     

Evaluation of a communication and stress management training programme for infertile couples

OBJECTIVE: This study evaluates a patient education programme focussed on improving communication and stress management skills among couples in fertility treatment. METHODS: In total, 37 couples completed the intervention. Two teachers conducted all the five courses offered. The effectiveness regarding communication and infertility-related stress was assessed by questionnaires immediately before (time T1) and after the intervention (time T2). Seeking of information and professional support was assessed at a 12-month follow-up (time T3); response rates were: T1, 93.2%; T2, 85.1%; T3, 74.3%. Data were compared at baseline (T1) and at the 12-month follow-up (T3) with a prospective cohort of Danish people in fertility treatment. RESULTS: There were no differences in infertility-related stress at base line between the two groups studied. We estimated the bi-directional changes in communication, e.g., changes from talking often to talking less frequently and vice versa. More intervention participants started to talk often with their partner about infertility and its treatment after the intervention compared to those who stopped to talk often. Women and men changed occurrence, frequency and content of communication with close other people. Among women marital benefit increased significantly. Infertility-related stress was not reduced significantly. Significantly more intervention participants than in the comparison group had contacted support groups, a psychologist and/or agencies for adoption at the 12-month follow-up. CONCLUSION: The intervention resulted in important perceived improvement in the participants' competence to actively manage changes in marital communication and in communication in different social arenas. PRACTICE IMPLICATIONS: We recommend fertility clinics to develop and evaluate different interventions for those fertility couples who ask for more psychosocial support.     

Chemokines and soluble adhesion molecules in renal transplant recipients with cytomegalovirus infection

Cytomegalovirus (CMV) infection is associated with leucocyte infiltration in various organs, which supports a role for chemokines and adhesion molecules in the pathogenesis of CMV infection. In a prospectively conducted study of renal transplant recipients, 10 patients with CMV disease, five patients with asymptomatic CMV infection and 10 patients who did not have any CMV infection were included. During CMV infection, and in particular during CMV disease, plasma levels of the chemokines IL-8, macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1) and the soluble adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and L-selectin increased and were positively correlated with the degree of CMV pp65 antigenaemia. Furthermore, a decrease in plasma levels of these chemokines and adhesion molecules was observed following ganciclovir therapy in the patients with CMV disease. This could suggest a role for these molecules in the pathogenesis of CMV infection.     

Apical and basolateral expression of Aquaporin-1 in transfected MDCK and LLC-PK cells and functional evaluation of their transcellular osmotic water permeabilities

 Aquaporin-1 is present in the apical and basolateral membranes in proximal tubules and descending limbs of Henlé’s loop. In order to be able to study the routing of Aquaporin-1 and the regulation of Aquaporin-1-mediated transcellular water flow, we stably transfected LLC-PK₁ and MDCK-HRS cell lines with an Aquaporin-1 expression construct. LLC-PK₁ clone 7 and MDCK clone K integrated two and one copies, respectively, which was reflected in the amount of Aquaporin-1 mRNA expressed in both clones. The Aquaporin-1 protein levels, however, were similar. In both clones, immuno-electronmicroscopy showed extensive labelling of Aquaporin-1 on the basolateral plasma membrane, endosomal vesicles and the apical plasma membrane, including the microvilli. To measure transcellular water permeation, a simple method was applied using phenol-red as a cell-impermeant marker of concentration. In contrast to the native cell lines, both clones revealed a high transcellular osmotic water permeability, which could not be influenced by forskolin add/3-isobutyl-1-methylxanthine (IBMX) or the phorbol ester 12-O-tetradecanoyl 13-acetate (TPA). After glutaraldehyde fixation, it was inhibitable by HgCl₂. These results indicate that targeting of Aquaporin-1 to the apical and basolateral plasma membrane is independent of cell type and show for the first time that water flow through a cultured epithelium can be blocked by mercurial compounds. Received: 9 October 1996 / Received after revision: 3 January 1997 / Accepted: 8 January 1997     

Faster activation of polymorphonuclear neutrophils in resistant mice during early innate response to Pseudomonas aeruginosa lung infection

Polymorphonuclear neutrophils (PMNs) are crucial for the outcome of Pseudomonas aeruginosa lung infection in patients with cystic fibrosis. We compared PMNs and inflammatory cytokines in the lungs and blood from susceptible BALB/c and resistant C3H/HeN mice 1 and 2 days after intratracheal challenge with alginate embedded P. aeruginosa. These parameters were correlated with the quantitative bacteriology and histopathology of the lungs. After challenge, the content of granulocyte colony-stimulating factor (G-CSF) and macrophage inflammatory protein-2 (MIP-2) was increased in the lungs and the sera and the percentage of PMNs was increased in the blood. However, 2 days after challenge the concentration of G-CSF and MIP-2 was higher in the lungs and sera of BALB/c mice. CD11b expression was higher on the PMNs of the C3H/HeN mice. The expression of CD62L on PMNs of both strains of mice was decreased 1 day after bacterial challenge, whereas the expression was increased after 2 days of challenge on PMNs of C3H/HeN mice only. These changes were accompanied by a more severe lung inflammation in BALB/c mice and faster clearance of the bacteria in C3H/HeN mice. In conclusion, the rapid early bacterial clearance in the lungs of C3H/HeN mice could be explained by faster activation of the PMNs, as indicated by the higher up-regulation of CD11b. The severe lung inflammation in BALB/c mice may be caused by the early higher content of G-CSF in the sera mobilizing PMNs from the bone marrow and the persistent chemotactic gradient provided by MIP-2 in the lungs.     

Adaptation of wild-type measles virus to CD46 receptor usage

Summary.  Vaccine strains of measles virus (MV) use CD46 as receptor and downregulate CD46 from the surface of infected cells. MVs isolated and passaged on B-lymphoid cells (wild-type MVs) seem to use another receptor and do not downregulate CD46. In the present study, we found that isolation of MV on human or marmoset B-lymphoid cells did not alter the MV haemagglutinin (H) protein relative to that in the patient. The wild-type isolates were adapted to the human epithelial HEp-2 cell line or the monkey fibroblast Vero cell line. All HEp-2 cell adapted viruses and 1 out of 4 Vero cell adapted viruses acquired the capacity to use CD46 as receptor, as measured by their ability to infect murine cells expressing human CD46. Adaptation to CD46 receptor usage was coupled to substitution of amino acid 481 of the MV H protein from asparagine to tyrosine but not to CD46 downregulation. The present study demonstrates that CD46 receptor usage can be induced by adaptation of wild-type MV to cells that do not express a wild-type receptor and suggests that a similar mechanism acted on the progenitor viruses of the present MV vaccine strains during their isolation and attenuation. Received June 5, 2000 Accepted October 5, 2000     

Blood pressure response to low level static contractions

Summary The present study re-examines the 15% MVC concept, i.e. the existence of a circulatory steady-state in low intensity static contractions below 15% of maximal voluntary contraction (MVC). Mean arterial blood pressure was studied during static endurance contractions of the elbow flexor and extensor muscles at forces corresponding to 10% and 40% MVC. Mean value for endurance time at 10% MVC was significantly longer for flexion [111.3 (SD 56.1) min] than for extension [18.1 (SD 7.5) min;n = 7]. At 40% MVC the difference in mean endurance time disappeared [2.3 (SD 0.7) min for elbow flexion and 2.3 (SD 0.7) min for elbow extension]. Mean arterial blood pressure exhibited a continuous and progressive increase during the 10% MVC contractions indicating that the 15% MVC concept would not appear to be valid. The terminal blood pressure value recorded at the point of exhaustion in the 10% MVC elbow extension experiment was identical to the peak pressure attained in the 40% MVC contraction. For the elbow flexors the terminal pressor response was slightly but significantly lower at 10% MVC [122.3 (SD 10.1) mmHg, 16.3 (SD 1.4) kPa] in comparison with 40% MVC [130.4 (SD 7.4) mmHg, 17.4 (SD 1.0) kPa]. When the circulation to the muscles was arrested just prior to the cessation of the contraction, blood pressure only partly recovered and remained elevated for as long as the occlusion persisted, indicating the level of pressure-raising muscle chemoreflexes. Based on blood pressure recordings obtained during the occlusion, it is suggested that the slight reduction in terminal pressor response seen in the 10% MVC elbow flexion experiment was due to a reduced chemoreflex drive characteristic of a slow twitch muscle group during prolonged low force contractions.     

Isolation ofPasteurella multocida in a patient with spontaneous peritonitis and liver cirrhosis

A case of spontaneous bacterial peritonitis caused by Pasteurella multocida in a 12 year old boy with previously undiscovered cirrhosis of the liver is reported. This case is discussed and related to eight published cases of spontaneous peritonitis caused by Pasteurella multocida in adults, seven with cirrhosis of the liver and/or alcohol abuse, and one with systemic lupus erythematosus complicated by membranoproliferative glomerulonephritis. It would appear that spontaneous bacterial peritonitis caused by Pasteurella multocida is not confined to adults with a history of alcohol abuse or cirrhosis of the liver, but can also affect children with non-alcoholic cirrhosis of the liver.     

Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa)

The classification of some of the extractable birch pollen antigens as allergens was established by crossed radioimmunoelectrophoresis (CRIE). In CRIE the major allergen (antigen 23) exhibited the strongest “radiostaining” and only a few other components of birch pollen extract were visibly radiostained. The major allergen and a preparation containing mainly the minor allergens, antigens 25 and 19, were isolated from a crude aqueous birch pollen extract by a combination of anion-exchange, size-exclusion, and chelate chromatography. Antigen 23 was purified to near homogeneity. The molecular weights and the pIs of antigens 23, 25, and 19 were determined to be 17,000 daltons, pI 5.25 (5.5, 5.0); 25,000 daltons, pI 5.0 (4.9, 5.4); and 29,000 daltons, pI 6.2 (5.4), respectively. The classification of antigen 23 as the major allergen in birch pollen was supported by results of RAST inhibition experiments, RAST screening, and skin prick testing.