The Journal of Experimental Medicine

Moehring, Thomas J.; Moehring, Joan M.; Kuchler, Robert J.; Solotorovsky, Morris

doi: 10.1084/jem.126.3.407pmid: 5340609

The response to diphtheria toxin of two sensitive cell lines, KB and HeLa, was investigated. Inhibition of the incorporation of radioactively labeled amino acids into protein was the earliest detectable effect of diphtheria toxin. It was observed that, during the period of intoxication, the cell membrane was morphologically intact and retained its semi-permeable character, although it was rendered fragile and more easily disrupted by mechanical manipulations than the normal cell. The transport of amino acids continued even after intoxicated cells had ceased to synthesize protein, and the levels accumulated were equal to those of control cells. It was observed that cultural conditions, age, and handling of cells affected their response to toxin. In early log phase cells subjected to a minimum of handling before application of the toxin, the normally observed latent period preceding detectable effects was reduced to 15 min for KB cells and 30 min for HeLa cells, shorter times than previously reported. The data are consistent with the hypothesis that diphtheria toxin enters susceptible cells, possibly by pinocytosis, and there acts upon cytoplasmic sites of protein synthesis. Footnotes Submitted: 19 March 1967

Mishell, Robert I.; Dutton, Richard W.

doi: 10.1084/jem.126.3.423pmid: 6034749

A culture system for cell suspensions from mouse spleens has been described. The system provides adequate conditions for in vitro immunization on initial exposure to heterologous erythrocytes. The in vitro response closely parallels that observed in vivo with respect to size, early kinetics, antigen dose, and the inhibitory effect of passive antibody. The response of cultured cells differs in two respects from that seen in vivo. There is an increase in the ability to discriminate between different varieties of homologous erythrocytes and the in vitro response does not appear to be limited by whatever mechanisms regulate the in vivo response. Footnotes Submitted: 3 May 1967

Dutton, Richard W.; Mishell, Robert I.

doi: 10.1084/jem.126.3.443pmid: 6034750

The role of proliferation in the development of 19S antibody-forming cells in the primary response has been investigated in an in vitro system. Spleen cell suspensions from normal, unimmunized mice were cultured in vitro in the presence of mammalian erythrocytes and the number of 19S hemolytic plaque-forming cells that arose 4 days later was measured. The hot pulse technique for the selective irradiation of those cells which synthesize DNA during a defined period of time has been described. The effect of such hot pulses administered at various times after the addition of antigen on the subsequent appearance of antibody-forming cells was determined. The results established that: ( a ) the onset of DNA synthesis does not start for approximately 24–32 hr after the addition of antigen, ( b ) essentially all the antibody-forming cells arise by cell division, and ( c ) different cell populations are involved in the response to two non-cross-reacting antigens. Footnotes Submitted: 3 May 1967

Streilein, J. W.; Billingham, R. E.

doi: 10.1084/jem.126.3.455pmid: 5341945

Rats have been shown to be capable of displaying the various kinds of delayed cutaneous hypersensitivity reactions attributable to transplantation immunity previously described in guinea pigs, hamsters, rabbits, dogs, and man. The rat is unlike most other species in that much larger numbers of lymphoid cells are needed to incite these cutaneous reactions. With direct, transfer, or normal lymphocyte transfer reactions, the cutaneous responses were greater when donor and recipient differed at the Ag-B histocompatibility locus than when donor and recipient shared the same Ag-B alleles. An experiment was performed in which adult rats of a genetically defined backcross population, resulting from matings between DA and (DA x Lewis) F 1 hybrid rats, were inoculated intradermally with lymph node cells from DA rats sensitized against tissues from Lewis-strain donors. Some of the R 2 animals gave a biphasic transfer reaction with peak reactivities occurring first at 48 hr and recurring at 96–120 hr, while the others lacked this second component. Hemagglutination tests revealed that the R 2 rats giving the biphasic response possessed the Ag-B,1 antigen, which is also present in Lewis rats, whereas rats which gave monophasic reactions were homozygous for the Ag-B,4 antigenic determinant which is present in the DA strain. This suggested that the recall flare at 96–120 hr reflects proliferative activity on the part of the inoculated cells confronted by the disparate Ag-B isoantigen in the host's dermis. Skin homografts from R 1 animals bearing the Ag-B,1 antigen were uniformly rejected by DA hosts in 11 days or less, while grafts from backcross animals homozygous for the Ag-B,4 antigen usually lived longer, being rejected in 9 to 27 days. Evidence is also presented which suggests that specific isoantibodies may act synergistically with immune lymphocytes to bring about cutaneous inflammatory reactions in the rat. Footnotes Submitted: 8 May 1967

Wannamaker, Lewis W.; Yasmineh, Walid

doi: 10.1084/jem.126.3.475pmid: 6034751

Streptococcal DNAse C is more resistant to heat inactivation than the A or B enzyme. DNAses A and C are indifferent to the bacterial ribonucleic acid inhibitor whereas the B enzyme is markedly inhibited. Prolonged digestion with relatively large amounts of DNAse B results in chemical and biological destruction of the inhibitor. Ribonuclease as well as deoxyribonuclease activity is associated with the B enzyme. Both activities require divalent cations and both are inhibited by bacterial ribonucleic acid. The ratios of the two activities are constant in various preparations and after partial heat inactivation. Mutual inhibition of the two activities can be demonstrated in mixed substrate systems. The evidence presented is consistent with the view that the B enzyme is a single nuclease which can attack both deoxyribonucleic and ribonucleic acids. Footnotes Submitted: 8 May 1967

Wannamaker, Lewis W.; Hayes, Barbara; Yasmineh, Walid

doi: 10.1084/jem.126.3.497pmid: 4962268

Preparations of streptococcal DNAse D with high specific activity and free of other streptococcal nucleases have been obtained by zone electrophoresis and column chromatography. Antisera prepared by injecting rabbits with such preparations specifically neutralize the activity of this enzyme. As with DNAse B, preparations of DNAse D regularly exhibit ribonuclease activity. For both B and D enzymes, the order of substrate preference is thymus DNA, yeast RNA, bacterial RNA; but the specific activity of the D enzyme is higher than that of the B enzyme with respect to thymus DNA and lower with respect to bacterial RNA. Both the deoxyribonuclease and the ribonuclease activities exhibited by preparations of both enzymes are inhibited by bacterial RNA, but approximately 100-fold greater concentrations of bacterial RNA are required to achieve inhibition of the deoxyribonuclease activity of the D enzyme equivalent to the inhibition of the B enzyme. The deoxyribonuclease activity of the D enzyme is also inhibited by yeast RNA, but even larger amounts are required. These observations indicate that the D enzyme is immunologically distinct from the other streptococcal nucleases and that it differs quantitatively from the B enzyme with respect to relative specific activities on different substrates and behavior in the presence of the bacterial ribonucleic acid inhibitor. Footnotes Submitted: 8 May 1967

Danes, B. Shannon; Bearn, Alexander G.

doi: 10.1084/jem.126.3.509pmid: 4962269

Clones of skin fibroblasts from normal individuals, patients with different mucopolysaccharidoses, and certain of their relatives have been examined for cellular metachromasia and cellular uronic acid. All the clones derived from affected individuals and heterozygous carriers in families with the autosomal forms of Hurler's syndrome showed marked metachromasia and increased cellular uronic acid. Since only one cell population was demonstrated in clones derived from heterozygous carriers, no evidence for autosomal inactivation was obtained. Clones derived from affected individuals with the X-linked recessive form of Hurler's syndrome contained uniform populations of metachromatic staining cells which demonstrated increased cellular uronic acid. Clones derived from the noncarrier fathers showed no cellular metachromasia or increased cellular uronic acid. Clones derived from the heterozygous mothers and sisters showed two populations both qualitatively and quantitatively. On the average, 72% of these clones were metachromatic and demonstrated an increased uronic acid content; 28% of the clones showed no metachromasia and the uronic acid content was similar to that found in normal individuals. The appearance of two distinct cell populations in clones derived from females heterozygous for the X-linked recessive form of Hurler's syndrome provides evidence in favor of the Lyon hypothesis. Footnotes Submitted: 10 May 1967

Parr, Earl L.; Schaedler, Russell W.; Hirsch, James G.

doi: 10.1084/jem.126.3.523pmid: 6034752

A chronic infiltration of polymorphonuclear leukocytes was invariably found in the infertile regions of uteri containing foreign bodies in conventional rats, germfree rats, mice, and rabbits. Polymorphonuclear leukocytes were never found in the fertile regions of these uteri. A foreign body in the uterus of the rat, and probably also the mouse, was associated with a bacterial infection which spread the inflammatory response throughout the horn containing the foreign body, and in the mouse occasionally into the control horn as well. No bacteria could be cultured from the rabbit uterine horn containing a foreign body. In the germfree rat, both the infiltration of polymorphonuclear leukocytes into the uterus and fertility were significantly different from that observed in the conventional rat. Whereas in the conventional rat the inflammation and infertility extended along the entire length of the uterine horn containing a small foreign body, in the germfree rat the inflammation and infertility were closely correlated to the position of the foreign body. As judged by measurements of lysozyme in the uterine lumens of rats and rabbits, polymorphonuclear leukocytes released their contents into solution in the uterine lumen. It is concluded that some substance derived from polymorphonuclear leukocytes may exert toxic effects on fertilized ova or on spermatozoa and thus be responsible for the infertility of uteri containing foreign bodies. Footnotes Submitted: 18 May 1967