Relationship between peach lipid transfer protein specific IgE levels and hypersensitivity to non-Rosaceae vegetable foods in patients allergic to lipid transfer protein.

BACKGROUND: Lipid transfer protein (LTP), the major allergen in Rosaceae in geographic areas where the prevalence of birch pollen allergy is low, is a widely cross-reacting pan-allergen, but the pattern of cross-reactivity to plant-derived foods botanically unrelated to Rosaceae shows much variability. OBJECTIVE: To examine the relationship between peach LTP specific IgE levels and cross-reactivity to several non-Rosaceae, plant-derived foods. METHODS: IgE specific for peach LTP was measured by enzyme-linked immunosorbent assay in serum samples from 40 patients with Rosaceae allergy monosensitized to LTP. Patients were considered monosensitized to this protein in the absence of sensitization to other cross-reacting, plant-derived foods as shown by negative skin prick test (SPT) results with both birch and mugwort pollen. SPTs with commercial extracts of walnut, hazelnut, peanut, celery, maize, rice, tomato, orange, and onion were performed to detect possible immunologic cross-reactivity to these foods. RESULTS: Patients with negative SPT results with non-Rosaceae foods showed significantly lower levels of IgE to peach LTP than patients showing skin reactivity to one or more non-Rosaceae foods (P < .001). A significant difference in specific IgE to peach LTP between patients with positive or negative SPT results was observed with each individual food (P < .001 in all cases). The level of IgE to peach LTP was strongly related to the number of positive SPT results with non-Rosaceae foods (r = 0.78; P < .001). Increasing levels of IgE to peach LTP were associated with skin reactivity to nuts (29/40 [72%]), peanut (27/40 [67%]), maize (16/39 [41%]), rice (14/39 [36%]), onion (13/37 [35%]), orange (9/32 [28%]), celery (11/40 [27%]), and tomato (8/39 [20%]). CONCLUSIONS: This study suggests that all allergenic determinants in LTP from vegetable foods other than peach cross-react with peach LTP determinants, whereas only some peach LTP epitopes cross-react with allergenic determinants on botanically unrelated, plant-derived foods. The high levels of IgE to peach LTP seem to reflect the presence of IgE targeting common allergenic determinants of LTP, causing cross-reactivity to botanically unrelated, vegetable foods. In LTP-allergic patients, increasing levels of IgE to peach LTP are paralleled by an increasing number of foods other than Rosaceae positive on SPT that cause clinical symptoms.     

A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive's tissues.

All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue's integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive's tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bps from FineFIX fixed tissues, against a maximum length of about 150 bps achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFix treated tissues were also compared with fresh tissues'ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.     

Effects of antimetastatic,antiinvasive and cytotoxic agents on the growth and spread of transplantable leukemias in mice

The effects of cytotoxic (cyclophosphamide, CCNU, GANU), antiinvasive (vincristine, vinblastine) and antimetastatic (ICRF-159, DM-COOK) agents have been compared in mice-bearing P388 and L1210 leukemias, and TLX5 lymphoma. The drugs tested increase the survival time of the treated mice in a manner consistent with a cytotoxic action in the case of cyclophosphamide, CCNU, GANU, vincristine and vinblastine. Leukemic infiltration of the brain after i.p. tumor implantation has been determined by bioassay of this organ, and is reduced by treatment with all of the drugs tested, with the exception of ICRF-159. DM-COOK appears to increase the life-span of the treated animals by the inhibition of leukemic spread rather than by a cytotoxic action. The marked cytotoxicity of vincristine and vinblastine is sufficient to account for failure to detect any antimetastatic effects of these agents. The lack of antidisseminative effect observed for ICRF-159 under the experimental conditions employed might be connected with the observation that the antimetastatic action of this drug on solid tumors is due to its effects on tumor blood vessels.     

A Cys634Gly substitution of the RET proto-oncogene in a family with recurrence of multiple endocrine neoplasia type 2A and cutaneous lichen amyloidosis

Germ-line mutations of the RET proto-oncogene, involving five cysteine residues at codons 609, 611, 618, 620 and 634, are associated with two variants of the inherited cancer syndrome multiple endocrine neoplasia type 2: type 2A and familial medullary thyroid carcinoma. The association of multiple endocrine neoplasia type 2A with the dermatological disorder cutaneous lichen amyloidosis has already been reported, and mutations in the Cys634 have been identified in different families. We describe here an additional pedigree in which multiple endocrine neoplasia type 2A and cutaneous lichen amyloidosis cosegregate. A Cys634Gly was identified by direct sequencing of the RET proto-oncogene exon 11 in the affected individuals. The mutation creates a new HaeIII site, and restriction analysis performed on all family members rules out the presence of the altered allele in two children and consequently the risk of developing thyroid tumors. These results emphasize the role of molecular analysis of the RET proto-oncogene in diagnosing presymptomatically those individuals at risk of inheriting the disease allele.     

Detection of clinical markers of sensitization to profilin in patients allergic to plant-derived foods

BACKGROUND: A proper classification of patients allergic to plant-derived foods is of pivotal importance because the clinical features of allergic reactions to fruits and vegetables depend on the nature and characteristics of proteins responsible for sensitization. However, in normal clinical settings this is presently impossible. OBJECTIVE: We sought to detect clinical markers of sensitization to profilin. METHODS: Seventy-one patients allergic to fruits and vegetables but not sensitized to lipid transfer protein or natural rubber latex were studied. Food allergy was ascertained on the basis of clinical history and positive skin prick test responses with fresh foods, commercial extracts, or both. Allergies to foods that had caused less than 2 adverse reactions were confirmed by means of open oral challenge. IgE reactivity to rBet v 1/rBet v 2 and to natural Phleum species profilin were detected. Moreover, IgE to the 30- to 40-kd and 60- to 90-kd birch pollen-enriched fractions, which also can be involved in cross-reactivity phenomena, were measured in sera from 52 patients by means of ELISA. RESULTS: On the basis of in vitro tests, 24, 18, and 25 patients turned out to be sensitized to Bet v 1, Bet v 2, or both, respectively. Four patients had negative test results for both allergens. Hypersensitivity to Bet v 2 was strongly associated with clinical allergy to citrus fruits (39% in patients monosensitized to Bet v 2 vs 4% in patients monosensitized to Bet v 1, P <.025), melon or watermelon (67% vs 0%, P <.001), banana (66% vs 8%, P <.001), and tomato (33% vs 0%, P <.05), whereas Bet v 1 sensitivity was associated with clinical allergy to apple (100% vs 39%, P <.001) and hazelnut (56% vs 0%, P <.001). The sensitivity of a history of allergy to gourd fruits, citrus fruits, tomato, banana, or a combination thereof as a means to detect profilin-hypersensitive patients was 85% (41/48). The specificity of an allergy to any of these fruits exceeded 85%, with positive predictive values ranging between 68% and 91%. CONCLUSION: In clinical settings in which laboratory investigations are not easily accessible, allergy to melon, watermelon, citrus fruits, tomato, and banana can be used as a marker of profilin hypersensitivity once a sensitization to natural rubber latex and lipid transfer protein is ruled out.     

Macrolide-resistance genes in clinical isolates of Streptococcus pyogenes

Macrolide-resistance genes were investigated in 103 macrolide-resistant strains of Streptococcus pyogenes, isolated from children with pharyngotonsillitis. The presence of mef(A), erm(B), and erm(TR) genes was detected by PCR. mef(A) was found in 48 out of 103 (46.6%) strains, whereas erm(B) was detected in 43 isolates (41.7%). All mef(A) strains showed a typical M phenotype (resistance to 14- and 15-membered macrolides, and sensitivity to lincosamides and streptogramin B), whereas erm(B) strains had the MLSB phenotype (resistance to macrolides, lincosamides, and streptogramin B antibiotics). erm(TR) was found in 10 strains, always together with other resistance genes. In seven cases erm(TR) was associated with erm(B), and three cases with mef(A). In two isolates with the M phenotype (1.9%), it was not possible to detect the presence of any of the three macrolide resistance genes tested. Inducible resistance to macrolides was shown for 24 out of the 53 MLSB strains. Analysis of macrorestriction fragment patterns by pulsed-field gel electrophoresis showed that erythromycin-resistant S. pyogenes are polyclonal, however each phenotype, MLSB and M, formed essentially homogeneous groups.     

Protocol for real-time PCR identification of anthrax spores from nasal swabs after broth enrichment

A mass-screening protocol for the diagnosis of anthrax from nasal swabs based on an enrichment step in liquid medium was devised. Incubation for growth was performed in autoclavable vials and racks which allow real-time PCR analysis of sterilized cultures. A dual-color PCR was set up with primers and probes for the chromosomal marker rpoB and the plasmid marker lef. Specific primer and probe sets were designed for the differentiation of Bacillus anthracis from B. cereus and for the differentiation of the Sterne vaccine strain from field isolates and the Ames strain, which was used in the recent anthrax bioterrorist attack. The present protocol thus combines the high specificity and sensitivity of real-time PCR with excellent biosafety and the low hands-on time necessary for the processing of large numbers of samples, which is extremely important during control programs involving the processing of large numbers of samples.     

Experimental therapy of systemic lupus erythematosus: the treatment of NZB/W mice with mouse soluble interferon-γ receptor inhibits the onset of glomerulonephritis

Female NZB/W F1 mice develop an autoimmune disease similar to human systemic lupus erythematosus (SLE), and ultimately die of glomerulonephritis. Starting at the age of 16 weeks NZB/W F1 mice were treated for a period of 19 weeks with soluble interferon-γ receptor (sIFN-γR), anti-IFN-γ monoclonal antibody (mAb) or IFN-γ. All mice treated with sIFN-γR or anti-IFN-γ mAb were alive 4 weeks after the treatment was discontinued, whereas 50% of mice died in the placebo groups and 78% of the mice died in the IFN-γ-treated group. Histologically, there was severe membrano-proliferative glomerulonephritis in IFN-γ- and placebo-treated mice, and minimal or no mesangioproliferative disease in mice receiving sIFN-γR or anti-IFN-γ mAb. The renal mononuclear infiltrate (T lymphocytes and monocytes), expression of major histocompatibility complex class II antigen and glomerular immunoglobulin and complement deposition were reduced in those mice. These data suggest that an IFN-γ inhibitor, such as the soluble IFN-γR, can be used for SLE therapy in the early stages of the disease.