A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.

Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops. Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure. In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection. Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge. Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA. Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella. Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection. Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion. Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates. Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.

A role for stem cell factor and c-kit in the murine intestinal tract secretory response to cholera toxin.

The role of stem cell factor (SCF) and its receptor (c-kit) in the intestinal secretory response to cholera toxin (CT) was investigated using a ligated intestinal loop model in mice having mutations in the dominant white spotting (W) locus and the steel (Sl) locus. W/Wv mice, which express an aberrant form of the c-kit protein, failed to give an intestinal secretory response after luminal CT challenge. In contrast, W/Wv mice and their control littermates had equivalent intestinal secretory responses to Escherichia coli heat-stable enterotoxin (STa). Sl/Sld mice, which express only a soluble truncated form of SCF, also gave a significantly reduced intestinal secretory response to CT when compared to the secretory response of their littermate controls. The unresponsiveness of W/Wv mice to CT was restricted to the intestinal tract since these mice had foot pad swelling responses to CT challenge that were equivalent to their littermate controls. Restoration of mast cells in W/Wv mice by bone marrow transplantation of control littermate bone marrow did not reverse the CT-unresponsiveness of the intestinal tract. Histological evaluation of the gastrointestinal tract from W/Wv mice showed a normal distribution of enterochromaffin cells (ECC). CT challenge of either ligated intestinal loops from C57B1/6 mice or a mouse intestinal epithelial cell line (MODE-K) resulted in elevated levels of mRNA for SCF. MODE-K cells exposed to CT also had enhanced expression of c-kit. Finally, fluid obtained from CT-challenged ligated intestinal loops from C57B1/6 mice contained significant levels of SCF. Taken together, the above results suggest that CT-induced intestinal secretory responses are dependent upon SCF-c-kit interactions. These interactions appear to be induced as a consequence of CT stimulation of the intestinal tract and may also play a role in the development or functionality of the enteric nervous system.

CV706, a prostate cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity.

Radiation is an effective means of treating localized prostate cancer. However, up to 40% of men with certain risk factors will develop biochemical failure 5 years after radiotherapy. CV706, a prostate cell-specific adenovirus variant, is currently in clinical trials for the treatment of recurrent organ-confined prostate cancer. We demonstrated previously that a single administration of CV706 at 5 x 10(8) particles/mm3 of tumor eliminated established tumors within 6 weeks in nude mouse xenografts (Rodriguez et al., Cancer Res. 57: 2559-2563, 1997). We now demonstrate that CV706-mediated cytotoxicity is synergistic with radiation. In vitro, addition of radiation to CV706 resulted in a synergistic increase of cytotoxicity toward the human prostate cancer cell line LNCaP and a significant increase of virus burst size, with no reduction in specificity of CV706-based cytopathogenicity for prostate cancer cells. In vivo, prostate-specific antigen (+) LNCaP xenografts of human prostate cancer were treated with CV706 (1 x 10(7) particles/mm3 of tumor), 10 Gy of single fraction local tumor radiation, or both. Tumor volumes of the group treated with CV706 or radiation was 97% or 120% of baseline 6 weeks after treatment. However, when the same dose of CV706 was followed 24 h later with the same dose of radiation, the tumor volume dropped to 4% of baseline at this time point and produced antitumor activity that was 6.7-fold greater than a predicted additive effect of CV706 and radiation. Histological analyses of tumors revealed that, compared with CV706 or radiation alone, combination treatment with two agents increased necrosis by 180% and 690%, apoptosis by 330% and 880%, and decreased blood vessel number by 1290% and 600%, respectively. Importantly, no increase in toxicity was observed after combined treatment when compared with CV706 or radiation alone. These data demonstrate that CV706 enhances the in vivo radioresponse of prostate tumors and support the clinical development of CV706 as a neoadjuvant agent with radiation for localized prostate cancer.

Heparin-induced thrombocytopenia and thrombosis: detection and specificity of a platelet-aggregating IgG.

A 46-year-old female who died as a result of thrombocytopenia associated with multiple arterial occlusions and septicemia while on heparin therapy was found to have a platelet-aggregating factor present in several plasma samples and in a sample of serum. This factor was subsequently shown to be an IgG with aggregating properties toward normal platelets that were enhanced by, but not dependent on, the presence of heparin. Further studies showed that heparin was unlikely to have acted as a hapten in initiating the IgG production but that its role was significant in aggravating the ensuing arterial thrombosis. The necessity of substitution of heparin with alternative anticoagulant/antithrombotic therapy to avoid the worst sequelae of this potentially catastrophic syndrome is discussed.

Comparison of the biochemical, immunologic and allergenic properties of vespid venoms collected in early and late summer.

The biochemical, immunologic and allergenic properties of yellow hornet (Vespula arenaria) and bald-faced hornet (Vespula maculata) venoms collected in early and late summer were compared. The phospholipase A content of both hornet venoms decreased in late summer while protease, hyaluronidase and acid phosphatase contents were unchanged. The antigenic and allergenic properties of the two venoms, as measured by their reaction with rabbit antisera and sera from insect-allergic patients, respectively, were unchanged. These results suggest no changes in venom properties during the summer which influence the allergic response to insect stings.

Phospholipase A2 in venom extracts from honey bees (Apis mellifera L.) of different ages.

We measured phospholipase A2 activity in the venom of worker honey bees (Apis mellifera L.) of known ages using chemical (titrimetric) and radioallergosorbent methods. The two techniques give similar results. Low levels of phospholipase A2 are present in the venom system at the time of eclosion. Phospholipase A2 activity in the venom increases steadily through the 10 days after eclosion. Maximal phospholipase A2 levels (about 40 micrograms phospholipase A2/venom sac) are maintained through the rest of the life of a worker bee in summer.

Decreased Toll-like receptor-5 (TLR-5) expression in the mucosa of ulcerative colitis patients.

OBJECTIVE: Although there is a convincing evidence supporting an important role for microorganisms in the pathogenesis of Inflammatory Bowel Disease (IBD) which comprises ulcerative colitis (UC) and Crohn's disease (CD), the specific mechanisms involved remain unclear. Toll-like receptors (TLR) recognize various molecules of microbiota including flagellin, the principal protein of motile comensal and pathogenic bacteria implicated in the pathogenesis of IBD. AIM: To investigate the expression of the TLR-5 receptors at the mRNA and protein levels in the mucosa of UC patients. MATERIALS AND METHODS: TLR-5 mRNA was quantified by the validated real-time PCR (QPCR) in mucosal biopsies of 99 UC patients and 34 control patients and TLR-5 protein was detected by immunohistochemistry (IHC) in 57 UC and 10 control patients. RESULTS: Significantly decreased TLR-5 gene expression at mRNA and protein level was found in the mucosa of patients with moderate and severe disease activity as compared to patients with low UC activity and control. TLR-5 immunoreactivity was found in the mucosa of UC patients and normal controls in the cytoplasm of enterocytes and at their basolateral domain. However, the intensity of the IHC reaction in specimens from UC patients was substantially lower than in control samples. CONCLUSION: The decreased expression of TLR-5 gene and protein in the mucosa of UC patients suggests that down-regulation of TLR-5 is probably caused by the increased number of ligand molecules in the proximity of epithelial cells in the inflamed tissue.

Overexpression of the fragile histidine triad (FHIT) gene in inflammatory bowel disease.

OBJECTIVE: FHIT gene encodes human diadenosine triphosphate hydrolase involved in the regulation of cell cycle and nucleotide metabolism and is a candidate tumor suppressor gene. AIM: To investigate expression of FHIT gene at the mRNA and protein levels in sporadic inflammatory bowel disease (IBD). MATERIALS AND METHODS: FHIT mRNA was quantified by the validated real-time PCR (QPCR) and FHIT protein was detected by immunohistochemistry (IHC) in mucosal biopsies of 139 ulcerative colitis (UC), 19 Crohn's disease (CD) and 37 control patients. RESULTS: Significant FHIT gene overexpression was found in 78% of active UC but not in CD. IHC showed comparable results to QPCR. CONCLUSION: The local up-regulation of FHIT gene and protein expression in active UC may represent an adequate response against inflammatory challenge of epithelial cell homeostasis and protect against DNA damage and cell cycle disturbances.

Determination of elemental carbon emission.

The studies on elemental carbon content in the atmospheric air, performed at the air monitoring station in Katowice (Poland), have revealed violations of allowable maximum average annual and diurnal concentrations. Elemental carbon is introduced into the atmosphere mainly as soot generated from combustion processes. This work presents the determination of elemental carbon in emission generated from coal combustion processes.

Further studies of patients with both honeybee- and yellow-jacket-venom-specific IgE.

Twenty-five sera from patients with high titers of both honeybee- and yellow-jacket-venom-specific IgE were analyzed in RAST inhibition experiments, using each venom as the coupling and inhibiting antigen. Eight sera had unique antibody activity with no cross-reactivity between yellow-jacket- and honeybee-venom-specific IgE. In 5 sera, the IgE antibody activity was directed at a major allergen in yellow jacket venom cross-reacting with a minor allergen in honeybee venom. Honeybee venom inhibited only the honeybee venom RAST; yellow jacket venom inhibited both the honeybee and the yellow jacket venom RAST. Five sera showed the opposite pattern with IgE antibody directed at a major allergen in honeybee venom cross-reacting with a minor allergen in yellow jacket venom. The fourth newly observed pattern was that of extensive IgE antibody cross-reaction found with 7 sera. Both honeybee venom and yellow jacket venom inhibited both the honeybee and yellow jacket venom RASTs. There were no clinical features such as age, sex, atopy or type of anaphylactic symptoms which could distinguish patients in each group. All but one patient had a history of multiple sting exposures. These data suggest multiple allergens in honeybee and yellow jacket venom with differing patterns of cross-reactivity and have important implications for proper venom immunotherapy.

Analysis of differing patterns of cross-reactivity of honeybee and yellow jacket venom-specific IgE: use of purified venom fractions.

Prior studies of sera from insect sting-allergic patients have analyzed the relationship of coexisting honeybee venom- and yellow jacket venom-specific IgE. Radioallergosorbent (RAST)-inhibition tests with these venoms revealed four different patterns of activity. In this present study, purified fractions prepared from these venoms were used to analyze these varying patterns. The hyaluronidases of yellow jacket venom and honeybee venom showed extensive cross-reaction. The phospholipases from these venoms showed minimal cross-reactivity; antigen 5 was restricted to yellow jacket venom. There was a high molecular weight component in yellow jacket venom with immunologic properties similar to honeybee venom acid phosphatase. Sera from individual patients showed quantitative and qualitative differences in the reactions to the major components of both venoms. The differences in the RAST-inhibition patterns in patients with elevated levels of both honeybee venom- and yellow jacket venom-specific IgE are accounted for by these differences as well as by differences in the cross-reactivity between the individual components.

Evaluation of RAST inhibition as a method for standardization of ragweed pollen extracts.

The RAST inhibition technique was evaluated as a method for standardization of ragweed pollen extracts. Seven different ragweed extracts were examined. There was no relationship between the RAST inhibition results and the commonly used parameters of extract potency including weight/volume, PNU and total protein measurements. Intradermal skin test end point titration was variable in different patients and could not correlated with RAST inhibition results. However, RAST inhibition potency did correlate with the antigen E content of the extract and this finding suggests that RAST inhibition might provide a method for better standardization of allergen extracts.

Allergenic potency of bee antigens measured by RAST inhibition.

The potency of various bee antigens including bee venom, several whole bee body extracts and fractions of bee venom was studied using the RAST inhibition method. As compared to whole bee body extract, bee venom was a much more potent inhibitor of both bee venom and whole body RAST, suggesting that venom has a greater capacity to bind specific bee IgE antibodies. Whole body extracts also varied substantially in their inhibiting activity. Phospholipase A and hyaluronidase were the most potent of the bee venom fractions suggesting their potential use as an assay for standardization of insect extracts.

Relationship of immunotherapy, seasonal pollen exposure and clinical response to serum concentrations of total IgE and ragweed-specific IgE.

The relationship of immunotherapy, seasonal pollen exposure and clinical results to serum total IgE and ragweed-specific IgE was studied in a group of 63 highly ragweed-sensitive individuals. Both immunological parameters rose after small doses of immunotherapy. After larger doses, IgE antibodies fell. Immunotherapy inhibited the seasonal rise in ragweed IgE antibodies, which occurs in nonimmunized patients. This decrease in IgE antibodies and inhibition of seasonal rise was associated with a good clinical response. This immunological pattern and concomitant successful clinical result appeared related to high-dose immunotherapy.

Stinging insect allergy: detection and clinical significance of venom IgE antibodies.

Venom-specific IgE antibodies in 109 sera from patients who had had immediate systemic allergic reactions following insect stings were measured by the radioallergosorbent (RAST) procedure. The majority of sera contained IgE antibodies to either bee, yellow jacket, or hornet venoms. Some sera had positive RAST reactions with 2 or 3 venoms, but others contained single venom-specific IgE antibodies. Of 24 patients who had large local reactions, the sera of 12 contained venom IgE antibodies. The RAST procedure provides an accurate means of documenting IgE-mediated allergic sensitivity to stinging insects.

Standardization of stinging insect extracts.

The measurement of IgE Ab to insects (bee etc.) can be accomplished using RAST-discs. This prevents the non-specific wheal and flare produced when skin tests in higher concentrations of either whole body or venom are used. It will also eliminate the possibility of a serious systemic reaction by direct skin testing. The in vitro neutralization of IgE antibody by pure fractions and or components employing the RAST disc method may well be a useful tool for standardization of allergens.

Allergy to honeybee body components: distinction from bee venom sensitivity.

Two patients with inhalant allergy to whole bee body components are described, documented by positive skin tests to whole bee body extract and the presence of serum bee body-specific IgE. Both had evidence of mild sensitivity to bee venom. RAST inhibition studies indicated that the IgE antibodies directed at whole bee body components and bee venom were distinct. These observations suggest that venom allergy from insect stings and bee body inhalant allergy are caused by different antigens.