A T cell-independent mechanism of macrophage activation by interferon-gamma

A primary interest in immunity to intracellular pathogenic microorganisms and tumors is to understand the mechanisms by which macrophages are activated for various functions. Two parameters of macrophage activation are the expression of the class II histocompatibility proteins or Ia molecules (1), and cytotoxic activity. The ability of T cells to induce these responses has been extensively documented and occurs via their secretion of interferon-gamma (IFN-gamma) after interaction with antigen (2-6). However, in a recent study using mice with the severe combined immunodeficiency (scid) mutation (7) which have no detectable T or B cell functions (7-9), we were surprised to find the induction of Ia expression on macrophages and the partial inhibition of bacterial growth after infection with Listeria monocytogenes (10). We have now utilized neutralizing monoclonal antibodies specific for murine IFN-gamma to investigate the mechanism of macrophage activation in scid mice. We show here that IFN-gamma can be produced by scid mice in the absence of lymphocyte-mediated immunity, and this IFN-gamma is important for macrophage activation during infection with Listeria. These results indicate the presence of an important T lymphocyte-independent mechanism of macrophage activation and IFN-gamma production in response to infection.     

Jejunal free autograft: analysis of complications and their resolution

Review of 101 patients who underwent 111 free jejunal autografts has demonstrated an absolute procedural failure rate of 13.5 percent. Salvage reconstruction with a second jejunum was successful in six of nine patients and one third-time jejunum was successful, giving an overall salvage rate of 70 percent. There were 33 patients experiencing pharyngocutaneous fistulas, 20 of whom had been previously irradiated. Of these patients, 15 experienced spontaneous closure and 9 others had successful surgical correction. The mortality rate was 5 percent. Eighty-three percent of patients were restored to adequate per oral alimentation. The jejunum, despite its relatively high complication rate, is an excellent method for pharyngoesophageal reconstruction, expeditiously providing return to function for patients with late-stage disease.     

An ultraviolet resonance Raman study of dehydrogenase enzymes and their interactions with coenzymes and substrates

Ultraviolet resonance Raman (UVRR) spectra, with 260-nm excitation, are reported for oxidized and reduced nicotinamide adenine dinucleotides (NAD+ and NADH, respectively). Corresponding spectra are reported for these coenzymes when bound to the enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and liver and yeast alcohol dehydrogenases (LADH and YADH). The observed differences between the coenzyme spectra are interpreted in terms of conformation, hydrogen bonding, and general environment polarity differences between bound and free coenzymes and between coenzymes bound to different enzymes. The possibility of adenine protonation is discussed. UVRR spectra with 220-nm excitation also are reported for holo- and apo-GAPDH (GAPDH-NAD+ and GAPDH alone, respectively). In contrast with the 260-nm spectra, these show only bands due to vibrations of aromatic amino acid residues of the protein. The binding of coenzyme to GAPDH has no significant effect on the aromatic amino acid bands observed. This result is discussed in the light of the known structural change of GAPDH on binding coenzyme. Finally, UVRR spectra with 240-nm excitation are reported for GAPDH and an enzyme-substrate intermediate of GAPDH. Perturbations are reported for tyrosine and tryptophan bands on forming the acyl enzyme.     

TGF-Beta inhibits the in vitro induction of lymphokine-activated killing activity

Summary Employing serum-free media, human peripheral blood mononuclear cells, and purified recombinant interleukin-2 (IL-2), conditions were observed in which the development of IL-2-driven cytotoxic activity was suppressed. The cytotoxic activity of such IL-2-generated lymphokine activated killing (LAK) was tested against natural killer-resistant cultured tumor cells (Daudi, Raji, and a glioma). LAK generation was inhibited by addition of some normal sera, normal platelets, or some tumor cells. Because recent reports have indicated that transforming growth factor-beta (TGF-beta)-like factors are often secreted by tumors and the acidic alpha granules of platelets and can be present in sera, we tested the effect of purified human TGF-beta on the activation of LAK. Our results indicated that TGF-beta is very suppressive for LAK induction, and can completely prevent both the IL-2-driven proliferation and cytotoxicity at concentrations as low as 5 ng/ml. Titrations of IL-2 and of TGF-beta indicated that the suppression is dose-dependent and can be avoided by employing higher levels of IL-2. It was also found that the suppressive effect of TGF-beta can be overcome by washing suppressed cell populations and further culture in low levels of IL-2. Collectively, these data indicate that TGF-beta can be a potent inhibitor of LAK generation under standard activation conditions, but that this effect is regulated by the relative level of IL-2 and may be overcome and/or reversed in vitro.     

Antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens

Simple methods for the generation, purification, and assay of antibodies to the alpha-subunit of insulin receptor from eggs of immunized hens have been described. Chicken antibodies against the alpha-subunit inhibit insulin binding to the receptor and stimulate glucose oxidation as well as autophosphorylation of the beta-subunit. Thus the properties of chicken antibodies are very similar to those of antibodies found in human autoimmune diseases and different from rabbit antibodies obtained against the same antigen.     

Comparative glucose and pentose utilization by a thermophilic Lactobacillus

Abstract A thermophilic Lactobacillus , isolate VB183, was tested for comparative glucose and pentose utilization. The possible adverse effect of high concentrations of metabolic end products on cell yield was lessened or eliminated by using low substrate concentrations. Possible growth due to intracellular reserve material, was counteracted by starving the cells for 90 min before being used as inoculant. Isolate VB183 exhibited a higher growth rate on glucose (μ= 1.08 h⁻¹) than on ribose (μ= 0.76 h⁻¹). L. salivarius was included as a pentose non-fermenting control and showed no significant difference between growth on basal medium with and without ribose. VB183 was also found to utilize l (+)-arabinose but not d (−)-arabinose, stressing the importance of the isomer of the sugars tested.     

Acute lethality after x-irradiation of Tribolium confusum adults

Recently emerged adults of Tribolium confusum, maintained at 30° in flour-yeast medium, were exposed to X-ray doses between 8 and 75 kR at dose rates of 225 or 1000 R/min. In all cases, there was no significant mortality for about 10 days, and all deaths attributable to the irradiation occurred during the next 10 to 15 days; after the lower doses, many insects survived this critical period and then lived on for many months. The relative dose-independence of the survival-time of decedents suggested that there is a specific mode of death, i.e., an as-yet-unrecognized acute lethal syndrome. Irradiated beetles incubated at 22° exhibited about twice as great a delay in the onset of mortality, and a mortality period with about twice the duration, as those at 30°, suggesting the necessity of a sequence of metabolic events in the development of lethality. Beetles on a cornstarch diet were at least as radioresistant as beetles on flour yeast; beetles at 22° were distinctly more sensitive than those kept at 30°. Older beetles, exposed 4 or 10 months after emergence, showed progressive increases in acute radiosensitivity. Implications of these findings for radiobiological investigations on adult insects are discussed.

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Lethalite aigüe apres irradiation aux rayons X des adultes de Tribolium Confusum

Résumé La plupart des insectes utilisés dans les expériences de radiobiologie ont une longévité relativement brève à l'état adulte. De ce fait, il est difficile ou impossible de distinguer la mortalité aigüe liée à des mécanismes spécifiques d'une diminution nonspécifique de la résistance à un stress.La longévité des adultes de Tribolium rend possible de séparer ces deux catégories de réponses, elle peut aussi permettre la mise en évidence des différents modes de léthalité aigüe.Des adultes récemment formés de Tribolium confusum, maintenus à 30° dans le milieu farine-levure, ont été exposés à des doses de rayons X de 8 à 75 kR aux taux de 225 ou 1000 R/min. Dans tous les cas, il n'y a pas eu de mortalité significative pendant 10 jours, et toutes les morts attribuables à l'irradiation ont eu lieu au cours des 10 à 15 jours suivants; après les doses les plus faibles, beaucoup d'insectes ont survécu à cette période critique et ont alors vécu de nombreux mois. L'indépendance relative par rapport à la dose de la durée de la survie des descendants suggère l'existence d'un mode spécifique de mortalité, ou d'un syndrome léthal aigu.Les insectes irradiés en élevage à 22° ont présenté un délai de début de mortalité environ le double et une durée de la période de mortalité environ double de ceux enregistrés à 30°. Ceci représente un Q₁₀ d'environ 2,3, qui suggère la nécessité d'une succession de phénomènes métaboliques dans le développement de la léthalité.Des insectes sur un régime à base d'amidon de maïs étaient au moins aussi radiorésistants que ceux sur farine et levure; les insectes à 22° furent sans conteste plus sensibles que ceux élevés à 30°. Des coléoptères plus âgés, exposés 4 ou 10 mois après leur formation, ont montré des augmentations progressives dans la radiosensibilité aigüe.Ces résultats ont deux conséquences importantes pour d'autres recherches radiobiologiques sur les insectes adultes. La mise en évidence de l'existence d'un syndrome léthal spécifique et de ses délais de manifestation devrait accélérer l'analyse des facteurs intervenant dans la mort des insectes irradiés; d'autre part dans toute expérimentation radiobiologique sur des insectes adultes, les imagos doivent être d'un âge uniforme et connu.

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This work was supported by U. S. Public Health Service, NIH Grant GM-10208. During 1964–1965, the senior author held a Special Fellowship (1-F3-GM23423), U. S. Public Health Service, at the Department of Zoology, University of Cambridge; he is indebted to Professor C. F. A. Pantin, F. R. S., and other members of the Department, and especially to Dr. George Salt, F. R. S., both for extending use of their facilities and for many illuminating discussions.     

Membrane biogenesis during B cell differentiation: most endoplasmic reticulum proteins are expressed coordinately

The induction of high-rate protein secretion entails increased biogenesis of secretory apparatus organelles. We examined the biogenesis of the secretory apparatus in the B cell line CH12 because it can be induced in vitro to secrete immunoglobulin (Ig). Upon stimulation with lipopolysaccharide (LPS), CH12 cells increased secretion of IgM 12-fold. This induced secretion was accompanied by preferential expansion of the ER and the Golgi complex. Three parameters of the rough ER changed: its area and volume increased 3.3- and 3.7-fold, respectively, and the density of membrane-bound ribosomes increased 3.5-fold. Similarly, the area of the Golgi stack increased 3.3-fold, and its volume increased 4.1-fold. These changes provide sufficient biosynthetic capacity to account for the increased secretory activity of CH12. Despite the large increase in IgM synthesis, and because of the expansion of the ER, the concentration of IgM within the ER changed less than twofold during the differentiation process. During the amplification of the rough ER, the expression of resident proteins changed according to one of two patterns. The majority (75%) of rough microsomal (RM) proteins increased in proportion to the increase in rough ER size. Included in this group were both lumenal proteins such as Ig binding protein (BiP), and membrane proteins such as ribophorins I and II. In addition, the expression of a minority (approximately 9%) of RM polypeptides increased preferentially, such that their abundance within the RM of secreting CH12 cells was increased. Thus, the expansion of ER during CH12 differentiation involves preferential increases in the abundance of a few resident proteins, superimposed upon proportional increases in most ER proteins.     

A nonselective cation channel activated by membrane deformation in oocytes of the ascidianBoltenia villosa

Summary Cell-attached patch clamp recordings from unfertilized oocytes of the ascidianBoltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. The estimated density of these stretch-activated channels is about 1.5/m², a figure equal to or greater than the density of known voltage-dependent channels in the oocyte. Ion substitution experiments done with combined whole-cell and attached patch recording, so absolute potentials are known, indicate that the channel passes Na⁺, Ca²⁺ and K⁺, but not Cl^–. The channel has at least two open and two closed states, with the rate constant that leaves the longer-lived closed state being the primary site of stretch sensitivity. External Ca²⁺ concentration affects channel kinetics: at low calcium levels, long openings predominate, whereas at high calcium virtually all openings are to the short-lived open state. In multiple channel patches, the response to a step change in suction is highly phasic, with channel open probability decreasing over several hundred milliseconds to a nonzero steady-state level after an initial rapid increase. This channel may play a role in the physiological response of cells of the early embryo to the membrane strains associated with morphogenetic events.