Sensitization of mice with wild-type and cold-adapted influenza virus variants: immune response to two H1N1 and H3N2 viruses

Two A strain influenza viruses, A/Hong Kong/123/77 (A/HK/123/77) (H1N1) and A/Queensland/6/72 (A/Qld/6/72) (H3N2), and the two cold-adapted reassortants which possess the surface antigens of these strains (CR35 and CR6, respectively) were tested for their ability both to induce primary cytotoxic T-cell (Tc cell) responses in mice and to sensitize mice for a second Tc cell response when challenged with a distantly related A strain virus, A/Shearwater/72 (H6N5). After intranasal inoculation, A/Qld/6/72 replicated to higher titers in the lung (1 to 2 log10 50% egg infective doses) than did A/HK/123/77 or either of the reassortants. A/Qld/6/72 induced higher Tc cell responses in the lung than did CR6, and both were more effective than either A/HK/123/77 or CR35 in this respect. When similar doses (10 or 10(3) hemagglutinin units) of each virus were injected intravenously into mice and the spleens were tested for Tc cell activity 6 days later, both A/Qld/6/72 and CR6 were ca. 100-fold better at inducing a primary Tc cell response than A/HK/123/77 or CR35. In contrast, the H1N1 and H3N2 viruses gave rather similar anti-hemagglutinin antibody titers (after intravenous injection) and delayed-type hypersensitivity reactions (after subcutaneous injection). If mice were primed with a low dose of these viruses (10(4) 50% egg infective doses intranasally), A/Qld/6/72 and CR6 were more effective than A/HK/123/77 or CR35 at sensitizing for a secondary Tc cell response when challenged with A/Shearwater/72, but if larger doses were given either intranasally (10(6) 50% egg infective doses) or intravenously (10 to 10(3) hemagglutinin units), all viruses sensitized the mice equally well, despite the fact the A/Shearwater/72 gives a poor primary Tc cell response in mice. Thus, the viral glycoprotein antigens can be important in determining the immunogenicity of the virus and, particularly, the class I antigen-restricted Tc cell response of the host.     

Partial purification and characterization of locust allatotropin I

Methanol extracts of locust brains, corpora cardiaca (CC), and suboesophageal ganglia (SOG) were separated by gradient and/or isocratic reverse-phase high-performance liquid chromatography (HPLC) and allatotropic activity monitored in the eluted fractions. A major peak of activity, separated by isocratic separation with 12% 2-propanol, designated allatotropin I, exhibited identical retention times in the three tissue extracts. Doseresponse curves of allatotropin I indicate similar content in brain and CC-equivalents, whereas optic lobes, similarly separated by isocratic HPLC, contain only one-tenth of this amount of allatotropin. Allatotropin I is resistant to boiling and is susceptible to tryptic and chymotryptic digestion. Methanol extracts of thoracic muscle, Malpighian tubules, fat body or ovaries, similarly prepared and boiled, did not exhibit allatotropic activity at high doses of tissue equivalents.     

Influenza virus-specific antibody-secreting cells in the murine lung during primary influenza virus infection.

An enzyme-linked immunosorbent plaque assay is described which can reliably enumerate influenza virus-specific antibody-secreting cells and exhibits specificity similar to that of the indirect enzyme-linked immunosorbent assay. The assay was used to characterize the development of specific antibody-secreting cells, principally within lung tissue, during primary murine influenza virus infection after intranasal inoculation. Cells secreting influenza virus-specific immunoglobulin M (IgM), IgG, and IgA were detected in greatest numbers in lung tissue, and the data presented indicated that the cells may have originated from specific B-cell precursors in lung tissue which are demonstratable in vitro. At 11 months after infection, cells secreting IgG and IgA were still present in lung tissue. Influenza virus-specific antibody-secreting cells were also detected in spleen tissue and blood. Antibody-secreting cells appeared earlier in spleen than in lung tissue and declined more rapidly in spleen tissue.     

Studies on Analogues of Succinic Semialdehyde as Substrates for Succinate Semialdehyde Dehydrogenase from Rat Brain

The methyl ester of succinic semialdehyde (SSA) was examined as a substrate for succinate semialdehyde dehydrogenase (SSADH) from rat brain. It was found that the ester can be oxidized by the enzyme. Values of Km for SSA-Me were higher than for those for SSA, and for this substrate the enzyme showed a substrate-dependent inhibition. This finding suggests that the carboxylate group of SSA is not essential in the process of inhibition of SSADH by the substrate. Cyclopropyl analogues of SSA, cis- and trans-1-formyl-cyclopropan-2-carboxylic acids, were also individually tested as substrates of SSADH. Only the trans isomer was found to be oxidized to the corresponding dicarboxylic acid; it inhibited the enzyme in the same range of concentrations as SSA. The above data suggest that, as for gamma-aminobutyric acid, SSA is present in an unfolded, transoid conformation at the active site of SSADH.     

Immunocytochemical localization of carbonic anhydrase in the spinal cords of normal and mutant (shiverer) adult mice with comparisons among fixation methods

The peroxidase-antiperoxidase technique was used for immunocytochemical localization of carbonic anhydrase in the mouse spinal cord to detect whether this antigen was normally present in myelinated fibers, in oligodendrocytes in both white and gray matter, and in astrocytes, and to determine where the carbonic anhydrase might be localized in the spinal cords of dysmyelinating mutant (shiverer) mice. The most favorable methods for treating tissue were: 1) immersion in formalin-ethanol-acetic acid followed by paraffin embedding, or 2) light fixation with paraformaldehyde and preparation of vibratome sections. Carnoy's solution, followed by paraffin embedding, extracted myelin from the tissue, while aqueous aldehydes, when used before paraffin embedding, reduced staining everywhere except at sites of compact myelin. The latter conclusion was based, in part, on the almost complete loss of this antigen from the shiverer cord, where compact myelin is known to be virtually absent but where membrane-bound carbonic anhydrase was demonstrated enzymatically. When the optimal methods were used with normal mouse cords, carbonic anhydrase was found throughout the white matter columns and in the oligodendrocytes in gray and white matter. The staining of the white matter was attributed to myelinated fibers because of the similarity in distribution to both a histological myelin stain and the immunocytochemical staining for myelin basic protein. In the mutant mice the oligodendrocyte cell bodies and processes, which were stained in all areas of the spinal cord, were particularly numerous at the periphery of the sections. In contrast to the oligodendrocytes, the fibrous astrocytes appeared to lack carbonic anhydrase, or to have lower than detectable levels, since the astrocyte marker, glial fibrillary acidic protein, had a very different distribution from that of carbonic anhydrase. Even finer localization was obtained in vibratome sections, where the antibody against carbonic anhydrase permitted visualization of the processes connecting oligodendrocytes to myelinated fibers in the normal adult spinal cord.     

The colon of Leucophaea maderae: fine structure and physiological features

The colon of L. maderae consists of a single columnar epithelium covered with a cuticle and of a musculo-connective sheath. The apical plasma membranes form a system of leaflets with numerous mitochondria inserted in association with microfilaments. Lateral plasma membranes are linked together by junctional complexes consisting of a zonula adherens and a long convoluted septate junction of the pleated type. In the basal region of the cell, numerous membrane infolds and scattered scalariform junctions with associated mitochondria are present. These cell specializations are typical of arthropod transporting organs, being distinctive features of ion and fluid transporting epithelia. The isolated colon exhibited a transepithelial electrical potential difference (PD) of about 100 mV, lumen side positive with respect to the haemolymph side. The PD was almost abolished by metabolic inhibitors, it was reduced by acetazolamide and SITS, and it was unaffected by ouabain. These effects suggest that HCO3- and Cl- are involved in the genesis of the PD, whereas Na+ is not directly responsible of the PD. Measurements of Na+ and Cl- fluxes across the colon wall confirm that Na+ moves following the PD across the tissue, while Cl- movement occurs against an electrochemical potential difference. The electrical profile of the epithelial cells is of the well type and it suggests that the primary or secondary active step for Cl- transport across the epithelium should be located at the mucosal border of the cell.