Development of an auxotrophic oral live Shigella flexneri vaccine

An oral live attenuated Shigella flexneri vaccine candidate strain was constructed by making it auxotrophic and dependent on aromatic metabolites not available in mammalian tissues. An aroD gene of Escherichia coli K12 strain NK 5131, inactivated by insertion in it of the Tn 10 transposon, was transduced using phage P1 into a virulent S. flexneri serotype Y strain (Sfl 1) isolated from a patient with bacillary dysentery. One of the transductant strains Sfl 114 was found to invade HeLa cells in vitro, to cause plaque formation in HeLa monolayers (i.e. maintain intracellular multiplication in vitro), but to be unable to cause keratoconjunctivitis in guinea-pig eyes. When the strain was fed to Macacca fascicularis monkeys it was well tolerated, excreted for 1-4 days, and found to elicit a local intestinal sIgA and serum IgA, IgM and IgG responses. Monkeys challenged with 100 ID50 dose (1 X 10(11) bacteria) of the virulent parent Sfl 1 strain were completely protected from development of diarrhoea. Coloscopy of the monkeys and the sampling of intestinal biopsies showed that the vaccine protected against the surface epithelial erosions and ulcerations seen in unimmunized monkeys. Killing of invading virulent shigellae apparently took place intracellularly in the mucosa suggesting that cellular immune mechanisms played a role in the elicited host defence. The constructed S. flexneri Sfl 114 strain has the properties of a promising shigella vaccine and will next be the subject of studies with human volunteers.     

Effects of low and high dose oral contraceptives on blood coagulation and thrombogenesis induced by vascular subendothelium exposed to flowing human blood.

We investigated the effect of oral contraceptives with low and high estrogen concentration on blood coagulation and thrombogenesis, induced by vascular subendothelium of rabbit aorta exposed to flowing human blood. Twenty healthy women intending to take oral contraceptives were studied [1] before drug ingestion (control), and subsequently during the intake of oral contraceptives with [2] low estrogen content (20 micrograms ethinyl estradiol and 150 micrograms desogestrel per day) and [3] high estrogen content (50 micrograms ethinyl estradiol and 125 micrograms desogestrel per day). All experiments were performed between day 17 and 21 of the menstrual cycle and drug effects were studied during the third tablet cycle. Deposition of fibrin, platelets and platelet thrombi on vascular subendothelium was tested at a defined blood flow and wall shear rate (10 ml/min, 650 s-1) and was quantified by morphometrical techniques. Treatment with the low and high dose contraceptive increased the plasma levels of ethinyl estradiol (728 +/- 139 and 1438 +/- 212 vs. 0 fmol/l [low and high dose vs. control], means +/- SEM, P less than 0.001) and fibrinogen (2.3 +/- 0.1 and 2.6 +/- 0.1 vs. 2.0 +/- 0.1 g/l, P less than 0.05); and decreased antithrombin III activity (95 +/- 3 and 92 +/- 3 vs. 101 +/- 3 %, P less than 0.05). Fibrin deposition on vascular subendothelium was enhanced by the high dose contraceptive only (47 +/- 4 vs. 35 +/- 4 % coverage of the subendothelial surface with fibrin, high dose vs. control, P less than 0.05). The subendothelial deposition of platelets and platelet thrombi was not changed by contraceptive treatment. These results indicate that treatment with high dose contraceptives leads to an increase of fibrin-subendothelial interactions, whereas low dose contraceptives do not significantly alter the blood-subendothelium interactions. observed in this ex vivo model of thrombogenesis.     

Cysticercosis of the brain. The value of computed tomography

Incidence and radiologic findings of neurocysticercosis were investigated in a series of 23,800 consecutive head examinations using computed tomography (CT). The condition was diagnosed in 168 cases (0.7%). The parenchymatous form was the most common presentation (96.3%), while the meningeal form corresponded to only 11.9 per cent of cases. These two forms coexisted in some cases. These findings reversed the knowledge on the condition based on conventional radiography. The different CT appearances in the brain are described and a new radiologic protocol for the CT evaluation of the condition is advocated, which includes a follow-up after a trial cure with Praziquantel in the presence of cysts not associated with suggestive brain calcifications. CT was more sensitive than conventional radiography in the differentiation between dead and living larvae, thus having an impact on the therapeutic management of the patients.     

Quantitative and comparative study of haematoporphyrin-derived photosensitizers on a murine tumour model

Eight commercially available HPD-photosensitizers intended for photodynamic therapy were tested in a murine tumour model with regard to their therapeutic efficacy. The regrowth delay of the fibrosarcoma SSK-2 on the mouse C3H, Neuherberg-line, was determined 3, 24, 48 and 72 h after injection of the drugs (dose: 9 mg kg^–1 body weight). The corresponding pharmacodynamics, as measured by regrowth delay, were approximated by an exponential function and the characterizing coefficients derived. These coefficients served to quantify the photodynamic properties of the drugs.The pharmacodynamics of five substances were compared with those obtained fluorometrically. The latter showed shorter decay constants than the therapy-correlated substances which indicates different metabolic behaviour of the therapeutic and diagnostically useful fluorescent components of haematoporphyrin-derived photosensitizers.     

Assessment of the potential in vivo genotoxicity of fluoranthene

Fluoranthene is a ubiquitous environmental pollutant. Althoughfluoranthene is mutagenic in bacterial and mammalian in vitrocell systems following metabolic activation by rat liver fraction,information on in vivo mutagenicity is lacking and studies ontumour initiating activity in mice are equivocal. In the presentstudy, the potential genetic hazard to man was assessed usingthe mouse bone marrow micronucleus and rat liver unscheduledDNA synthesis test systems. Fluoranthene did not show any evidenceof genotoxicity in either of the in vivo assays following acuteoral administration at levels of up to 2000 mg/kg b.w. ¹To whom correspondence should be addressed     

Antibody response and protection against challenge in mice vaccinated intraperitoneally with a live aroA O4-O9 hybrid Salmonella dublin strain.

An auxotrophic Salmonella dublin (O9,12) strain, SL5631, with a deletion affecting gene aroA, was made into a partial diploid expressing the rfb (O-antigen-repeat-unit-specifying) gene cluster of Salmonella typhimurium (O4,12). By use of O4- and O9-specific antisera in indirect immunofluorescence assays, the resulting hybrid SL7103 was shown to express both the O4- and O9-antigen epitopes in the same bacterium. Qualitative and quantitative sugar analyses by gas-liquid chromatography on peralditol acetates of phenol-water-extracted lipopolysaccharides showed that the S. dublin and S. typhimurium repeating units (estimated on the basis of their tyvelose and abequose contents, respectively) were present in approximately equimolar amounts. The SL7103 hybrid auxotroph was avirulent when given intraperitoneally to NMRI mice in a dose of 10(8) CFU and elicited a protective immunity against intraperitoneal challenge with either virulent S. dublin (50% lethal dose of ca. 1.5 x 10(4) CFU versus < 1 x 10(1) CFU in nonimmunized mice) or virulent S. typhimurium (50% lethal dose of ca. 1 x 10(5) versus < 1 x 10(1) CFU in nonimmunized mice). Compared with the protection elicited in homologous systems (S. dublin SL5631 against S. dublin and S. typhimurium SL1479 against S. typhimurium), the protective efficacy of the hybrid was reduced approximately 70-fold against S. dublin challenge and 100-fold against S. typhimurium challenge. Vaccination with S. typhimurium SL1479 conferred no protection against S. dublin challenge, and vaccination with S. dublin SL5631 conferred no protection against S. typhimurium challenge. The protection elicited by the hybrid strain SL7103 is supposed to be mainly a consequence of serum antibodies directed against the immunodominant O4 and O9 epitopes.     

A randomized trial comparing combination electron-beam radiation and chemotherapy with topical therapy in the initial treatment of mycosis fungoides

Mycosis fungoides is a T-cell lymphoma that arises in the skin and progresses at highly variable rates. Nonradomized studies have suggested that early aggressive therapy may improve the prognosis in this usually fatal disease. We studied 103 patients with mycosis fungoides, who, after complete staging, were randomly assigned to receive either combination therapy, consisting of 3000 cGy of electron-beam radiation to the skin combined with parenteral chemotherapy with cyclophosphamide, doxorubicin, etoposide, and vincristine (n = 52) or sequential topical treatment (n = 51). The prognostic factors were well balanced in the two groups. Combined therapy produced considerable toxicity: 12 patients required hospitalization for fever and transient neutropenia, 5 had congestive heart failure, and 2 were later found to have acute nonlymphocytic leukemia. Patients receiving combined therapy had a significantly higher rate of complete response, documented by biopsy, than patients receiving conservative therapy (38 percent vs. 18 percent; P = 0.032). After a median follow-up of 75 months, however, there was no significant difference between the treatment groups in disease-free or overall survival. We conclude that early aggressive therapy with radiation and chemotherapy does not improve the prognosis for patients with mycosis fungoides as compared with conservative treatment beginning with sequential topical therapies.     

Connections of the caudal ventrolateral medullary reticular formation in the cat brainstem

 A region of the caudal ventrolateral medullary reticular formation (CVLM) participates in baroreceptor, vestibulosympathetic, and somatosympathetic reflexes; the adjacent retroambigual area is involved in generating respiratory-related activity and is essential for control of the upper airway during vocalization. However, little is known about the connections of the CVLM in the cat. In order to determine the locations of terminations of CVLM neurons, the anterograde tracers Phaseolus vulgaris leucoagglutinin and tetramethylrhodamine dextran amine were injected into this region. These injections produced a dense concentration of labeled axons throughout the lateral medullary reticular formation (lateral tegmental field), including the retrofacial nucleus and nucleus ambiguus, regions of the rostral ventrolateral medulla, the lateral and ventrolateral aspects of the hypoglossal nucleus, nucleus intercalatus, and the facial nucleus. A smaller number of labeled axons were located in the medial, lateral, and commissural subnuclei of nucleus tractus solitarius, the A5 region of the pontine reticular formation, the ventral and medial portions of the spinal and motor trigeminal nuclei, locus coeruleus, and the parabrachial nucleus. We confirmed the projection from the CVLM to both the rostral ventrolateral medulla and lateral tegmental field using retrograde tracing. Injections of biotinylated dextran amine or Fluorogold into these regions resulted in retrogradely labeled cell bodies in the CVLM. However, the neurons projecting to the lateral tegmental field were located mainly dorsal to those projecting to the rostral ventrolateral medulla, suggesting that these neurons form two groups, possibly with different inputs. Injections of retrograde tracers into the lateral tegmental field and rostral ventrolateral medulla also produced labeled cell bodies in other regions, including the medial and inferior vestibular nuclei and nucleus solitarius. These data are consistent with the view that the CVLM of the cat is a multifunctional area that regulates blood pressure, produces vocalization, affects the shape of the oral cavity, and elicits contraction of particular facial muscles. Received: 18 February 1997 / Accepted: 27 March 1997