Immunologic and nonimmunologic responsiveness in ragweed-sensitized dogs

The relationship between airway responsiveness to inhaled antigen and histamine, immunologic release of lung histamine, immunologic responsiveness of skin, and specific immunoglobulin E (IgE) antibodies were examined in 11 inbred allergic dogs immunized with extracts of ragweed and grass and 5 nonimmunized control dogs from the same colony. Airway responsiveness to antigen and histamine was characterized by the doses that increased the airflow resistance of the total respiratory system to twice the control values (ED200). Highly significant correlations were found between airway responsiveness and cutaneous responsiveness to antigen and other immunologic characteristics (e.g., IgE and histamine released from lung by inhaled antigen) in all dogs. In ragweed-sensitized dogs, there was an inverse correlation between immunologic responsiveness (reflected by the cutaneous response to antigen and histamine released from lung by inhaled antigen) and nonimmunologic responsiveness of airways (histamine ED200: r = 0.73, P less than 0.05 and r = 0.75, P less than 0.01, respectively). Antigen ED200 was also correlated with histamine release from lung after antigen inhalation (r = 0.74; P less than 0.01). We conclude that airway reactions to inhaled antigen in allergic dogs are dependent not only on immunologic factors but also on the degree of nonimmunologic airway responsiveness to histamine and that these factors are correlated inversely.     

Cell-free processing and segregation of insulin precursors

The biosynthesis, segregation, and processing of preproinsulin (116 amino acids) was investigated to determine the mechanism(s) by which it is translocated across the endoplasmic reticulum membrane. Islet mRNA was translated in the wheat germ cell-free system, and at various times during preproinsulin synthesis, puromycin was added, followed by addition of microsomal membranes. Neither processing of preproinsulin nor translocation of proinsulin into microsomal membranes occurred in the presence of puromycin. Synchronization of preproinsulin translation by addition of 7-methylguanosine 5'-phosphate enabled the timing of preproinsulin synthesis and proinsulin (91 amino acids) segregation into microsomal membranes to be determined. Membrane binding occurs when about 60 amino acids have been polymerized, i.e. prior to the completion of the polypeptide chain. The binding of signal recognition particle to the nascent signal is demonstrated to be an absolute requirement for translocation and processing of preproinsulin. The results indicate that segregation and processing of preproinsulin are co-translational events; no evidence for a post-translational mechanism was found. Furthermore, this work, together with similar studies, suggests that presecretory polypeptides must be synthesized as part of a precursor with a minimum size of 60-80 amino acids in order to effect membrane binding and translocation of the polypeptide chain within the intracisternal space of the endoplasmic reticulum.     

Phagocytosis is coupled to the formation of phagosome-associated podosomes and a transient disruption of podosomes in human macrophages

Phagocytosis consists in ingestion and digestion of large particles, a process strictly dependent on actin re-organization. Using synchronized phagocytosis of IgG-coated latex beads (IgG-LB), zymosan or serum opsonized-zymosan, we report the formation of actin structures on both phagocytic cups and closed phagosomes in human macrophages. Their lifespan, size, protein composition and organization are similar to podosomes. Thus, we called these actin structures phagosome-associated podosomes (PAPs). Concomitantly to the formation of PAPs, a transient disruption of podosomes occurred at the ventral face of macrophages. Similarly to podosomes, which are targeted by vesicles containing proteases, the presence of PAPs correlated with the maturation of phagosomes into phagolysosomes. The ingestion of LB without IgG did not trigger PAPs formation, did not lead to podosome disruption and maturation to phagolysosomes, suggesting that these events are linked together. Although similar to podosomes, we found that PAPs differed by being resistant to the Arp2/3 inhibitor CK666. Thus, we describe a podosome subtype which forms on phagosomes where it probably serves several tasks of this multifunctional structure.     

Simple and selective high-performance liquid chromatographic method for estimating plasma quinidine levels

A reversed-phase, high-performance liquid chromatographic method employing fluorescence detection is described for the rapid quantification of plasma levels of quinidine, dihydroquinidine and 3-hydroxyquinidine. It involves protein precipitation with acetonitrile followed by direct injection of the supernatant into the chromatograph. For the preparation of plasma standards, pure 3-hydroxyquinidine was isolated from human urine by a simplified thin-layer chromatographic procedure. The mobile phase for the chromatography was a mixture of 1.5 mM aqueous phosphoric acid and acetonitrile (90:10) at a flow-rate of 2 ml/min. The intra-assay coefficient of variation for the assay of quinidine and 3-hydroxyquinidine over the concentration range 2.5–20 μmole/l was < 1% for both. Interassay coefficients of variation for quinidine (10 μmole/l) and 3-hydroxyquinidine (5 μmole/l) were 3.5% and 4.0% with detection limits of 50 and 25 μmole/l respectively. The method correlated well (r² = 0.96) with an independently developed gas—liquid chromatographic—nitrogen detection assay for quinidine which also possessed a high degree of precision. (Intra-assay coefficient of variation 3.6% at 20 μmole/l). As expected, comparison of the high-performance liquid chromatographic assay with a published protein precipitation—fluorescence assay showed poor correlation (r² = 0.78).     

Gypsy moth parasitoids in the declining outbreak in Lithuania

A 3 year study was conducted on the parasitoids of gypsy moth larvae in two reducing outbreak areas in Lithuania. Overall parasitism of 25.0 ± 2.0% in the first post-culmination year was significantly lower than the 36.3 ± 1.4 and 35.2 ± 1.4% parasitism in the two subsequent years. When analysed in terms of the life stage at which the host was collected, the total parasitism over 3 years was constantly increasing from 3.1 ± 0.8 in the first to 72.5 ± 2.9% in the sixth instar. Parasetigena silvestris R.-D. dominated causing 48.7 ± 1.5% parasitism and 16.7 ± 0.6% larval mortality preferably in late instars. Phobocampe disparis Vier. contributed to 21.9 ± 1.2% parasitism and 7.5 ± 0.5% mortality recovering from early instar larvae. Meteorus pulchricornis Wes. parasitized 4.3 ± 0.6% gypsy moth larvae causing 1.5 ± 0.2% mortality and a few Apanteles species provided 2.8 ± 0.6% parasitism and 1.0 ± 0.2% mortality. The gypsy moth in Lithuania was reported to act as host for Rogas sp. (Hym., Braconidae) and Siphona boreata Mes. (Dipt., Tachinidae).     

Identification of HLA-B44 subtypes associated with extended MHC haplotypes

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.     

Functional competency of T cell antigen receptors in human thymus

The T cell antigen receptor is likely to play a role in both positive and negative selection in the thymus. Three populations of thymocytes can be distinguished by the level of expression of the CD3-alpha/beta-chain heterodimer of the T cell antigen receptor (CD3/Ti alpha/beta) complex. Cells which fail to express these receptors or express low levels of receptors are contained in a population of thymocytes which express low levels of the CD5 antigen and are predominantly CD4+/CD8+. Thus, these cells appear to be relatively immature phenotypically. In contrast, the cells which express high levels of CD3/Ti alpha/beta co-express high levels of CD5 and are predominantly contained in the more mature single positive cells which express either CD4 or CD8. With the calcium-sensitive dye, Indo-1, and immunofluorescence, we demonstrated that, despite the relative phenotypic immaturity of cells which express low levels of CD3/Ti alpha/beta, these antigen receptors are able to mediate transmembrane signaling when stimulated with CD3 monoclonal antibodies. Although increases in calcium were observed in these CD3/Ti alpha/beta-low expressing cells in response to anti-CD3, no proliferative response was observed, even in the presence of phorbol myristate acetate. Proliferative responses were observed in the more mature cells which express high levels of CD3/Ti alpha/beta. These results suggest that, rather than a defect in the functional capability of the antigen receptor complex to mediate transmembrane signaling events, cellular responses to signals generated by the antigen receptor may differ at various stages of thymocyte development.     

The study of a French family with two duplicated C4A haplotypes

Summary The finding of two duplicated C4A haplotypes in a normal French family led to a detailed study of their C4 polymorphism. The father had an extremely rare A^*6A^*11, B^* QO haplotype inherited by all of his children and the mother had the more common A^*3A^*2, B^*QO haplotype. Two HLA identical daughters only have four C4A alleles. The father's A11 allotype expresses Ch: 1 (Chido) rather than Rg:1 (Rodgers) and represents a new Ch phenotype Ch: 1,-2,-3,-4,-5,-6. In order to clarify the genetic background in this unusual family, DNA studies of restriction fragment length polymorphisms (RFLPs) were undertake. The father's rare haplotype, which expresses two C4A allotypes, results from a long and a short C4 gene normally associated with the A^*6, B^*1 that also exhibits the BglII RFLP. As it travels in an extended MHC haplotype HLA A2, B57 (17), C2^*C, BF^*S, DR7 that is most frequently associated with A^*6, B^*1, we postulate that the short C4B has been converted in the chain region to a C4A gene which produces a C4A protein. This report of a short C4A gene is the first example in the complex polymorphism of C4.