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A murine pulmonary model was used to study the mucosal immune response to Shigella flexneri serotype 2a infection. Inoculation of BALB/cJ mice with shigellae via the intranasal route resulted in bacterial invasion of bronchial and alveolar epithelia with concomitant development of acute suppurative bronchiolitis and subsequent development of lethal pneumonia. The pathology of pulmonary lesions resembled the colitis that characterizes shigellosis in humans and primates. Significant protection against a lethal dose of S. flexneri 2a was observed in mice previously infected with two sublethal doses of the homologous strain. Immunity against lethal challenge was associated with decreased bacterial invasion of the mucosal epithelium. Over the course of two sublethal challenges, which constituted primary and secondary immunizations, mice developed pulmonary and serum immunoglobulin G and A antibody recognizing both lipopolysaccharide and invasion plasmid antigens IpaB and IpaC. Immune mice and naive control mice differed in lung lavage cytokine levels following lethal challenge. Immune mice developed significantly elevated levels of pulmonary gamma interferon within 6 h of challenge, while naive control mice developed elevated levels of this cytokine later during the initial 24-h period. Both groups had elevated levels of gamma interferon during the 24- to 48-h period of infection. Both groups also had elevated levels of tumor necrosis factor alpha within 6 h of challenge, but the control mice had significantly higher levels at the 48- and 72-h time points. Elevated levels of interleukin-4 were observed only in immunized mice. This cytokine appeared within 24 h and receded between 48 and 72 h. Fluorescence-activated cell sorter analysis of lung parenchymal cells showed that both groups experienced an initial influx of monocytes, but the proportion of this cell type began to recede in immunized mice after 48 h of infection, while peak levels were maintained in the control animals. These studies suggest that elements of local B lymphocyte activity, as well as Th1 and Th2 lymphocyte activity, may contribute to the survival of immune mice after intranasal challenge with shigellae.

Chloroquine (CQ), which is the primary drug for treatment and prophylaxis against malaria, has become ineffective because of the high prevalence of CQ-resistant P. falciparum parasites, but resistant parasites exposed to a Ca(II) channel blocker become as susceptible to CQ as sensitive parasites. A reversed-phase liquid chromatographic method is described for simultaneously determining CQ, its metabolites desethyl-CQ and bisdesethyl-CQ, the Ca(II) channel blocker verapamil, and the tiapamil analogue N-(3,4-dimethoxyphenethyl)- N-methyl-2-(naphthyl)-m-dithiane-2-propylamine hydrochloride. The analytes were separated by gradient elution with acetonitrile and 1-heptane sulfonic acid; the detector wavelength was 232 nm. The mean recovery from spiked plasma was 100.1 ± 2.28 (SD). Within-day retention times were reproducible to within ± 0.03 SD of mean values and the lower limit of detection was about 2 ng of each analyte.

This study used the guinea pig keratoconjunctivitis model to examine the importance of route of administration (mucosal versus parenteral), frequency and timing of immunization (primary versus boosting immunization), and form of antigen given (live attenuated vaccine strain versus O-antigen-protein conjugate) on the production of protective immunity against Shigella infection. Since local immune response to the lipopolysaccharide (LPS) O-antigen of Shigella spp. is thought to be important for protection against disease, O-antigen-specific antibody-secreting cells (ASC) in the spleen and regional lymph nodes of immunized animals were measured by using an ELISPOT assay. Results indicated that protective efficacy was associated with a strong O-antigen-specific ASC response, particularly in the superficial ventral cervical lymph nodes draining the conjunctivae. In naive animals, a strong ASC response in the cervical lymph nodes and protection against challenge were detected only in animals that received a mucosal immunization. Protection in these animals was increased by a boosting mucosal immunization. While parenteral immunization alone with an O-antigen-protein conjugate vaccine did not protect naive animals against challenge, a combined parenteral-mucosal regimen elicited enhanced protection without the addition of a boosting immunization. Although O-antigen-specific serum immunoglobulin A titers were significantly higher in animals receiving a mucosal immunization, there was no apparent correlation between levels of serum antibody and protection against disease.

The effects of two different invasive strains (TML and SL 1027) of Salmonella tryphimurium on Na and Cl transport in the absence of an electrochemical gradient were measured across rabbit ileal mucosa in vitro. Only strain TML elicits ileal fluid secretion in vivo. Net Na transport is negligible and net Cl secretion occurs across mucosae invaded by TML. In contrast, mucosae invaded by SL 1027 show absorption of both Na and Cl, as occurs in normal ileum. The luminal addition of glucose stimulates Na absorption without affecting Cl transport across ileum invaded by either strain. Theophylline stimulates Na transport across TML-invaded mucosa, causing net secretion, and enhances Cl secretion. Across SL 1027-invaded tissues, theophylline reverses spontaneous ion absorption, causing secretion of both Na and Cl. The data suggest that stimulation of active Cl secretion combined with inhibition of spontaneous Na absorption may account for the diarrhea of salmonella enteritis. However, invasion of the mucosa alone by S. typhimurium is not necessarily associated with major alterations in ion transport, suggesting that a factor, in addition to invasion, is necessary for fluid secretion. The effects of theophylline are compatible with strain TML exerting submaximal stimulation of cyclic adenosine monophosphate activity or involving another metabolic pathway which also inhibits spontaneous Na absorption and stimulates Cl secretion.

A 140-megadalton plasmid (pWR110), which has previously been associated with virulence in Shigella flexneri, was transferred to Escherichia coli K-12. Segments of S. flexneri chromosomal material were then transferred to the plamid-bearing K-12 strains. The virulence of these transconjugant hybrids was assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test. A K-12 strain which harbored only pWR110 invaded HeLa cells, produced minimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test. Plasmid-containing K-12 hybrids which had incorporated various shigella chromosomal regions gave differential reactions in the rabbit ileal loops and in the Sereny test. Analysis of these transconjugants indicated that three regions were linked with virulent phenotypes. These included the his region (when the genes responsible for O-antigen synthesis were cotransferred) and the kcp locus (linked to the lac-gal region). Either of these chromosomal regions was sufficient to allow invasion of the rabbit ileal mucosa. In addition to both of these regions, another shigella chromosomal segment linked to the arg and mtl loci was necessary for fluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus, derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transfer of a large plasmid and three chromosomal segments from S. flexneri, appeared to contain the necessary determinants for full pathogenicity in a variety of laboratory models.

The shedding of epidermis in this species, as in most amphibia, is periodic. A single outer layer of cells separates nearly simultaneously over the whole body; two hours to ten days may be required for the process. In the first three months of laboratory existence the average frequency of molting was 9 per cent. In nature the frequency was only 2 per cent; this increased during the life in confinement in five months up to 25 per cent. Death in the laboratory was usually preceded by numerous molts. The time of a molt is not normally determined by any one of a number of ordinary external conditions which were modified; but nevertheless several means were found of inducing molting. These were, in order of their efficacy: bandaging with gauze on body or tail, wrapping the body with thread, stitching the body wall, applying hot water, and transplanting skin. Molting frequencies up to 98 per cent may thus be obtained. Since molting is periodic and simultaneous, it is evidently coordinated. Experimental induction requires a latent period of six to ninety-six hours, depending upon the stimulus. Recent skin grafts molt with the host, so that the coordination is probably chemical. The primary significance of molting in all groups of animals is possibly the getting rid of unautolyzable waste materials, i.e., worn epithelial structures.

SummaryActive immunization with parenteral formalin-inactivated EMC virus or with oral living-attenuated EMC virus protets mice against lethal oral challenge with virulent virus and prevents subsequent viral shedding in the feces. Similar results can be obtained by passively immunizing mice with rabbit anti-EMC antiserum. Coproantibody was recovered only from orally immunized mice, and was thus not correlated with resistance to fecal shedding of virus.

It is perhaps not generally known that certain species of small rodents detach portions of the tail in a seemingly voluntary manner when they are seized by this member. Among the mouse-like rodents of California such an autotomy of the tail appears to be most typically exhibited by the pocket-mice (Perognathus). It is the general practice of the writers, as probably of most breeders of mice, to pick up their animals by the tail. This appendage constitutes a convenient handle by which a mouse may be lifted, commonly relieving one of the risk of being bitten. But such a procedure is hazardous in the case of Perognathus. By a sudden gyratory movement of the body, the tail is likely to be severed at some point in its length, allowing the animal to make its escape and giving the pursuer something of a handicap in the race. The whole performance is so suggestive of what occurs in many lizards that the question naturally arose in our minds: Can it be possible that autotomy, in the one case, as in the other, is followed by regeneration? This of course was not to be expected in a mammal, but the experiment seemed at least worth trying. In any case, the act and its consequences were thought to be worthy of a brief consideration. The species upon which most of these observations were conducted was a rather large pocket-mouse, Perognathus fallax fallax Merriam, which is very common in the neighborhood of La Jolla. Despite the frequency of autotomy in this species, we soon found that all individuals cannot be depended upon to "perform" when desired. Those, in particular, which have been kept captive for some time seem to lose the tendency. More

Salmonella typhi 5076-IC, which contains a plasmid that encodes the form I antigen of Shigella sonnei and which expresses S. typhi 9 and 12 and S. sonnei form I antigens, was used to immunize rabbits via chronically isolated ileal loops. Intestinal immunoglobulin A activity was detected against S. typhi, S. sonnei form I, and S. typhi strain 5076-IC. Thus S. typhi 5076-IC can effectively elicit mucosal immunoglobulin A to both S. typhi and S. sonnei.

Two methods are in general use for the evaluation of qualities. The paper suggests an alternative technique which may be employed, and advances evidence to show its superiority over the other methods. The practical application of the technique is illustrated by reference to metallurgical problems and to the assessment of the corrosivity of cutting oil emulsions.