Effect of a perfluorochemical emulsion on the radiation response of BA1112 rhabdomyosarcomas

The effect of treatment with a perfluorochemical emulsion (Fluosol DA, 20%), carbogen, or the combination of these two agents on the radiation response of BA1112 tumors in WAG/rij rats was examined. Fluosol and carbogen as single agents had only small effects on the tumor cell survival curve. The combination of Fluosol plus carbogen had a larger effect on tumor cell survival, reducing the hypoxic fraction of the tumor from 23 to 1.6%. The amount of sensitization was a function of the Fluosol dose, with maximal augmentation of the radiation response obtained at doses of 7.5-15 ml/kg. Carbogen pretreatments ranging from 5 to 60 min in duration all had similar effects on tumor radiosensitivity. The effect of the perfluorochemical emulsion plus carbogen on the survival of irradiated tumor cells appears to reflect changes in tumor oxygenation, rather than cytotoxic or immunological effects, since the perfluorochemical emulsion (with or without carbogen) had no effect on the viability of cells in unirradiated tumors. These experiments extend previous studies by ourselves and others using mouse tumors to show that the combination of a perfluorochemical emulsion and carbogen breathing can also increase the radiation response of a nonimmunogenic rat tumor.     

Effects of Fluosol DA 20% and carbogen on the radioresponse of SCK tumors and skin of A/J mice

The effect of Fluosol DA 20%, an emulsion of perfluorochemicals, in combination with carbogen (95% O2 and 5% CO2) breathing on the response of mouse tumors to radiation was studied. When A/J mice bearing SCK tumors in the right hind limb were injected iv with Fluosol DA 20% at 12 ml/kg and exposed to carbogen for 1 h before and during the irradiation of tumors, the response of tumors to a single dose of X irradiation was significantly enhanced. The dose modification factors for growth delay and cure of SCK tumors were 2.10 +/- 0.01 (SE) and 1.86 +/- 0.18 (SE), respectively. Such a treatment slightly increased the radiation-induced skin damage by a factor of 1.17 +/- 0.02 (SE), resulting in a therapeutic gain of 1.79 +/- 0.01 (SE) for the growth delay and 1.59 +/- 0.09 (SE) for the curability. Carbogen breathing alone also increased the response of tumor and skin to radiation, but it was far less effective than the combination of Fluosol DA 20% and carbogen breathing. It was concluded that iv injection of Fluosol DA 20% in conjunction with carbogen breathing significantly increased the O2 transport to hypoxic areas in the SCK tumors and thus significantly enhanced the tumoricidal effect of radiation on SCK tumors.     

Humoral immune responses among mucosal and cutaneous leishmaniasis patients caused by Leishmania braziliensis

Mucosal leishmaniasis is arguably the most morbid sequelae of cutaneous leishmaniasis. The importance of early diagnosis for effective therapy, coupled with the difficulty of diagnosing the disease parasitologically, prompted this investigation of humoral immune markers of mucosal disease. Promastigote soluble antigens of Leishmania braziliensis, isolated from cutaneous and mucosal lesions, were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis; antigens were identified by immunoblotting with parasite-specific IgG antibody-positive sera of patients with mucosal disease (n = 18) and cutaneous disease (n = 23). For antigens of the cutaneous parasite WR 2095, mucosal sera generally reacted intensely to antigens of 75, 66, and 45 kDa and weakly to 48-50-kDa antigens, whereas cutaneous sera generally detected weakly the first 3 antigens and intensely the latter doublet. The data suggest that the transition from the cutaneous antigenic profile to a mucosal antigenic profile could be used to predict mucosal disease in approximately half of mucosal patients. An additional finding was that antibodies present in the sera of patients with mucosal disease labeled a 66-kDa peptide of normal human lip mucosa more intensely than did cutaneous sera. Autoimmune processes stimulated by the reaction of IgG, originally directed against the 66-kDa of L. braziliensis, to the 66-kDa antigen of mucosal tissue may contribute to the clinical presentation of mucosal leishmaniasis.     

Induction of immunity against hepatitis B virus surface antigen by intranasal DNA vaccination using a cationic emulsion as a mucosal gene carrier

Delivery of DNA vaccines to airway mucosa would be an ideal method for mucosal immunization. However, there have been few reports of a suitable gene delivery system. In this study we used a cationic emulsion to immunize mice via the intranasal route with pCMV-S coding for Hepatitis B virus surface antigen (HBsAg). Complexing pCMV-S with a cationic emulsion dramatically enhanced HBsAg expression in both nasal tissue and lung, and was associated with increases in the levels of HBs-specific Abs in serum and mucosal fluids, of cytotoxic T lymphocytes (CTL) in the spleen and cervical and iliac lymph nodes, and of delayed-type hypersensitivity (DTH) against HBsAg. In contrast, very weak humoral and cellular immunities were observed following immunization with naked DNA. In support of these observations, a higher proliferative response of spleenocytes was detected in the group immunized with the emulsion/pCMV-S complex than in the group immunized with naked pCMV-S. These findings may facilitate development of an emulsion-mediated gene vaccination technique for use against intracellular pathogens that invade mucosal surfaces.     

Stimulation of mucosal immune response following oral administration of enterotoxigenic Escherichia coli fimbriae (CFA/I) entrapped in liposomes in conjunction with inactivated whole-cell Vibrio cholerae vaccine

In this study, we have searched for an effective mucosal vaccine. An oral enterotoxigenic E. coli vaccine containing colonization factor antigen (CFA/I) associated with inactivated whole-cell V. cholerae vaccine (WCV) has been tested for safety and immunogenicity in animals. Five groups of animals were used. The results showed the following: (a) vaccine containing CFA/I antigen entrapped in liposomes and associated with WCV (batch C) had increased titers of specific antibodies to CFA/I antigen in 15 to 18 (83.3%) animals; (b) specific Peyer's patches (PP), lymph nodes (LN) and spleen (SPL) lymphocytes proliferation was detected following in vitro restimulation with CFA/I antigen or WCV. This response gradually increased to the highest value by the 35th postimmunization day. Moreover, lower PP, LN and spleen (SPL) proliferation was observed in rabbits receiving soluble CFA/I antigen (S-CFA/I) or free liposomes (F-L) alone; (c) adhesion of E. coli H10407 strain labelled with 3H-leucine in immunized and control animals revealed the following local effects: (i) protection of rabbit intestinal mucosa against virulent E. coli cells; (ii) inhibition of adhesion of ETEC bacteria to intestinal mucosa and (iii) significantly faster release of E. coli H 10407 strain labelled with 3H-leucine from the intestinal tract of immunized animals. The histopathological and electron microscope findings confirmed the above results. The experimental results point out an efficient protection against infection with E. coli strains (ETEC), after mucosal vaccination with CFA/I antigen entrapped in liposomes associated with inactivated whole-cell Vibrio cholerae as immunological adjuvant.     

Mucosal antibody responses of colonized cattle to Escherichia coli O157-secreted proteins, flagellin, outer membrane proteins and lipopolysaccharide

The aim of this work was to characterize adaptive mucosal immune responses to Escherichia coli O157:H7 at the principal site of colonization in the bovine species. Following experimental infection, extracts from terminal rectum mucosal samples were tested for IgA antibodies by immunoblotting against different bacterial antigens including: whole-cell E. coli O157:H7 with and without proteinase treatment, outer membrane and cytoplasmic preparations, secreted protein supernatants and purified E. coli O157 lipopolysaccharide and H7 flagellin. Lipopolysaccharide and H7 flagellin preparations were also used to coat enzyme-linked immunosorbent assay plates to determine mucosal IgG1 and IgA antibody titers. In this work, evidence is presented of strong local IgA immune responses induced following infection at the bovine terminal rectal mucosa directed against multiple antigens including type III secretion-dependent proteins, O157 lipopolysaccharide, H7 flagellin and OmpC.     

Cecocolitis in immunodeficient mice associated with an enteroinvasive lactose negative E. coli

Infection with an atypical (lactose-negative) E. coli was associated with increased mortality rates in a colony of triple immune deficient N:NIH(S) III mice. Affected mice were lethargic and exhibited perianal fecal staining. Slight-to-moderate thickening of the wall of the cecum and colon was found on necropsy examination. Microscopic examination revealed segmental hyperplasia of the cecal and colonic mucosa with clusters of gram negative bacteria on the surface and within the cytoplasm of mucosal epithelial cells. Scattered foci of epithelial invasion and hyperplasia were observed in the colons of C57B1/6N-nunu mice after per os inoculation with the atypical E. coli. Immunocompetant mice housed in the same room as the N:NIH(S) III's remained healthy and exhibited no gross or microscopic lesions in spite of infection.     

The PIM-2 Kinase Is an Essential Component of the Ultraviolet Damage Response That Acts Upstream to E2F-1 and ATM

The oncogenic nature ascribed to the PIM-2 kinase relies mostly on phosphorylation of substrates that act as pro-survival/anti-apoptotic factors. Nevertheless, pro-survival effects can also result from activating DNA repair mechanisms following damage. In this study, we addressed the possibility that PIM-2 plays a role in the cellular response to UV damage, an issue that has never been addressed before. We found that in U2OS cells, PIM-2 expression and activity increased upon exposure to UVC radiation (2–50 mJ/cm²), and Pim-2-silenced cells were significantly more sensitive to UV radiation. Overexpression of PIM-2 accelerated removal of UV-induced DNA lesions over time, reduced γH2AX accumulation in damaged cells, and rendered these cells significantly more viable following UV radiation. The protective effect of PIM-2 was mediated by increased E2F-1 and activated ATM levels. Silencing E2F-1 reduced the protective effect of PIM-2, whereas inhibiting ATM activity abrogated this protective effect, irrespective of E2F-1 levels. The results obtained in this study place PIM-2 upstream to E2F-1 and ATM in the UV-induced DNA damage response.     

Papillomavirus pseudovirus: a novel vaccine to induce mucosal and systemic cytotoxic T-lymphocyte responses

Intestinal mucosa is a portal for many infectious pathogens. Systemic immunization, in general, does not induce a cytotoxic T-lymphocyte (CTL) response at the mucosal surface. Because papillomavirus (PV) naturally infects mucosa and skin, we determined whether PV pseudovirus, i.e., PV-like particles in which unrelated DNA plasmids are packaged, could generate specific mucosal immunity. We found that the pseudovirus that encoded the lymphocytic choriomeningitis virus gp33 epitope induced a stronger CTL response than a DNA vaccine (plasmid) encoding the same epitope given systemically. The virus-like particles that were used to make the pseudoviruses provided an adjuvant effect for induction of CTLs by the DNA vaccine. The PV pseudovirus pseudoinfected mucosal and systemic lymphoid tissues when administered orally. Oral immunization with the pseudovirus encoding human PV type 16 mutant E7 induced mucosal and systemic CTL responses. In comparison, a DNA vaccine encoding E7, when given orally, did not induce a CTL response in intestinal mucosal lymphoid tissue. Further, oral immunization with the human PV pseudovirus encoding E7 protected mice against mucosal challenge with an E7-expressing bovine PV pseudovirus. Thus, PV pseudovirus can be used as a novel vaccine to induce mucosal and systemic CTL responses.     

Trehalose dimycolate enhances survival of fission neutron-irradiated mice and Klebsiella pneumoniae-challenged irradiated mice

The survival of B6D2F1 female mice exposed to lethal doses of fission neutron radiation is increased when trehalose dimycolate (TDM) preparations are given either 1 h after exposure or 1 day before exposure to radiation. TDM in an emulsion of squalene, Tween 80, and saline was the most effective formulation for increasing the 30-day survival of mice when given 1 day before (90%) or 1 h after (88%) exposure to radiation. An aqueous suspension of a synthetic analog of TDM was less effective at increasing 30-day survival (60%) when given 1 day prior to radiation exposure and not effective when given 1 h after radiation. Mice receiving a sublethal dose (3.5 Gy) of fission neutron radiation and either the TDM emulsion or synthetic TDM 1 h after irradiation were substantially more resistant to challenge with 10, 100, 1000, or 5000 times the LD50/30 dose of Klebsiella pneumoniae than untreated mice.     

Effects of emulsified perfluorochemicals on growth and ultrastructure of microbial cells in culture

Summary The effects of emulsified perfluorochemicals (PFCs) and some of their constituents on growth and ultrastructure of microbial cells in culture have been studied. Growth rate ofE. coli was inhibited by stem emulsion from the proprietary formulation, Fluosol-DA 20%, and also by a combination of the Pluronic F-68 surfactant and yolk phospholipid emulsion stabilizer. Yeast growth was also inhibited by Fluosol stem emulsion and by pluronic alone. Electron microscopical examination of thin sections of yeast cells following culture in perfluorodecalin emulsion revealed cytoplasmic vacuolation and other ultrastructural perturbations resembling those reported previously in mammalian cells cultured with emulsified PFCs; no comparable structural changes inE. coli were observed.     

Altered colonizing ability for mouse large intestine of a surface mutant of a human faecal isolate of Escherichia coli

Escherichia coli F-17 Sr a human faecal isolate, is resistant to the T-series of bacteriophages (i.e. T2 to T7). A T2-sensitive mutant of E. coli F-17 Sr was isolated following acriflavin treatment. This mutant, E. coli F-17 Sr Ts was found to be sensitive to the entire T-series of phages. E. coli F-17 Sr and E. coli F-17 Sr Ts did not differ quantitatively in total LPS content. However, analysis of LPS revealed that a large fraction of E. coli F-17 Sr Ts was devoid of O-side-chains. This accounted for the sensitivity of this strain to bacteriophages T3, T4, and T7. In addition, E. coli F-17 Sr Ts contained only about half the amount of capsular material contained by E. coli F-17 Sr accounting for the sensitivity of the mutant to bacteriophages T2, T5, and T6. Although the two strains colonized equally well when fed individually to streptomycin-treated mice, when fed simultaneously to streptomycin-treated mice, E. coli F-17 Sr Ts colonized at a level of about 1 x 10(8) cells (g faeces)-1, whereas E. coli F-17 Sr colonized at only 1 x 10(4) cells (g faeces)-1. These studies suggest that bacterial cell surface components modulate the large intestine colonizing ability of E. coli F-17 Sr in the mouse large intestine.     

Prevention of non-steroidal anti-inflammatory agents induced acute gastric mucosal lesions by carbonic anhydrase inhibitors. An endoscopic study

The present paper studies the effect of acetazolamide, an inhibitor of carbonic anhydrase, on acute gastric mucosal damage induced by non-steroidal anti-inflammatory drugs. The study was performed on healthy male subjects. The drugs tested were aspirin (1.5 g/day), indomethacin (75 mg/day), phenylbutazone (600 mg/day) and ibuprofen (600 mg/day) given for 7 days in 3 divided doses. Each drug was given to 5 cases in two separate periods, during which they were given acetazolamide 20 mg/kg/day or placebo in random order. Dyspeptic symptoms were evaluated. Endoscopy was performed before, and 3 and 7 days after NOSAC administration. Gastric mucosal lesions were evaluated according to the scale proposed by Lanza (J. Clin. Pharmacol., 24: 1984, 89) and the severity of the lesions was calculated. All drugs tested produced dyspeptic symptoms and acute mucosal damage of the gastric mucosa. Inhibition of gastric mucosa carbonic anhydrase by acetazolamide cessated promptly dyspeptic symptoms and reduced significantly the number and severity of drug-associated mucosal lesions.     

丙线照射所致大鼠胃粘膜易损性及其与内源性PGs和血栓素A_2的关系

本文观察了丙线照射大鼠胃粘膜的易损性及其与由源性PG_s和血检素A_2的关系。结果表明:丙线1500rad局部照射后28天,大鼠对牛磺胆酸所致胃粘膜坏死易损性明显加重,预先给予外源性PGE_2则可抑制这一现象;进一步采用放射免疫方法测定胃粘膜PG_s等的含量发现,照射后组织PGE和PGI_2含量明显降低,而血栓素A_2含量则明显升高,PGI_2/血栓素A_2,比值下降。这些结果说明,丙线照射可使大鼠胃粘膜的易损性明显加重,而内源性PGE和PGI_2含量的降低以及血栓素A_2含量的升高是照射造成易损性的主要原因之一。     

Determination of the radioprotective effects of topical applications of MEA, WR-2721, and N-acetylcysteine on murine skin

Topical applications of MEA (beta-mercaptoethylamine or cysteamine), WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid], and N-acetylcysteine (NAC) were tested for their ability to protect the normal skin of the hind legs of mice against acute and late damage from single doses of 137Cs radiation. No significant protection was observed with either WR-2721 or NAC. MEA was shown to offer significant protection against acute skin damage in both buffered and unbuffered forms, but no significant protection against late contraction. The use of topical MEA on unanesthetized animals breathing carbogen (95% O2, 5% CO2) appears to give an enhanced level of radioprotection over that shown for anesthetized, air-breathing animals.     

Plasma progesterone and LH concentrations in ewes after injection of an analogue of prostaglandin F-2alpha

Plasma progesterone and LH concentrations were monitored throughout a natural oestrous cycle in 12 Clun Forest ewes and compared to those following treatment with a single i.m. injection of 100 microng ICI 80,996, an analogoue of prostaglandin F-2alpha, given during the luteal phase of the cycle. After injection of the analogue there was a high degree of synchrony in the return of oestrus (440 +/- 1-9 h; mean +/- S.E.M.) and the timing of the LH peak (48-5 +/- 2-0 h) from injection. There were no significant differences in the plasma progesterone concentrations or in the height and duration of the preovulatory LH peak between control and treatment cycles. The technique offers the possibility of controlled ovulation in the ewe.     

The binding of zinc to the soluble proteins of intestinal mucosa in winter flounder (Pseudopleuronectes americanus).

1. The binding of Zn2+ to soluble proteins of intestinal mucosa of winter flounder was examined using an equilibrium dialysis technique. 2. There appeared to be more than one binding system present for Zn2+ in the mucosal cytosol. 3. It required four times the normal endogenous Zn2+ level found in the mucosal cytosol to saturate the highest affinity (K1 = 2.42 x 10(7] binding system. 4. Of 10 metals tested Cu2+ was the only one which interfered with Zn2+ binding to the mucosal cytosol proteins. 5. It is postulated that binding proteins in the mucosal cytosol of winter flounder may play a role in the transport of Zn2+.     

An unexpected effect of hyperthermia on the expression of X-ray damage in mouse skin

The interaction between hyperthermia and X irradiation in the expression of injury to skin was investigated in the tail of adult mice. The X-ray treatments when given alone resulted in skin reactions which ranged in severity from "no observable gross injury" to "moist desquamation over most of the tail," the peak reaction occurring at approximately 20 days. When hyperthermia was given alone, the maximal reaction observed was "foci of moist desquamation, accompanied by severe erythema and edema" which, in contrast to the radiation response, peaked 1 to 2 days after treatment. For the combined treatments, hyperthermia at a temperature between 43.0 and 44.5 degrees C for 30 min was given either 3, 6, 9, or 10 days after X irradiation. When the interval was 3 days, there appeared to be no interaction between the treatments. As the interval was lengthened, so that hyperthermia was given 6 or more days after irradiation, i.e., within 7 days of the time of appearance of gross radiation injury, the severity of the observed skin reaction was greater than the individual responses following either treatment given alone. Using a 9-day interval, it could be seen that both the thermal and radiation reactions were enhanced in a dose-dependent manner. The peak times for each reaction were not significantly altered by the additional treatment. The results are discussed with reference to possible modes of interaction between X irradiation and hyperthermia in an in vivo system.     

Hepatic glutathione S-transferase activity in mice: effects of Avy/-genotype, obesity, lindane treatment, and sex

Phenotypically distinct but genetically identical obese mottled yellow Avy/a and lean pseudoagouti Avy/a sibling mice and their congeneic black a/a littermates provide an experimental system for distinguishing phenotypic effects from genotypic effects in the expression of the genotype at the organismic level. Hepatic glutathione S-transferase activity in obese yellow Avy/a (YS X VY) F-1 hybrid female mice was only about 66% of that found in their lean black a/a sisters. This decreased enzyme activity was not a direct effect of the Avy/a genotype but was associated with the obesity of the yellow mice since the enzyme activity in lean pseudoagouti Avy/a female siblings was similar to that found in the black a/a mice. Long-term feeding of 160 ppm lindane in the diet decreased the enzyme activity in all phenotypes but did not eliminate the difference between the obese yellow and lean pseudoagouti and black mice. Interpretation of the available data suggests that no direct relationship exists between the level of hepatic glutathione S-transferase activity and the enhancement of tumor formation in yellow Avy/a mice. Several inbred mouse strains and F-1 hybrids were also screened for this enzyme activity. No strain differences were found but sex differences within different inbred strains were not uniform. In the AE and YS strains and their F-1 hybrid enzyme activity was higher in female than in males. In contrast, BALB/c and VY strain males had higher enzyme activity than the corresponding females.