In vitro and inhibition of anaphylactic reactions 1-methyl-3-isobutylxanthine]

1-methyl-3-isobutylxanthine (MIBX), a potent antiphosphodiesterase drug, inhibited the anaphylactic reactions both in vitro and in vivo: anaphylactic histamine release from the isolated mast cells of rats, anaphylactic contracture of the isolated trachea of guinea pigs, passive cutaneous anaphylaxis (PCA) in mice and anaphylactic bronchial constriction (ABC) in guinea pigs. MIBX inhibited the anaphylactic reaction of the trachea, PCA and ABC more than the corresponding reactions induced by histamine.     

Dissociation of experimental allergic encephalomyelitis protective effect and allergic side reactions in tolerization with neuroantigen

Administration of autoantigens under conditions that induce type 2 immunity frequently leads to protection from T cell-mediated autoimmune diseases. Such treatments, however, are inherently linked to the induction of IgG1 Abs and to the risk of triggering anaphylactic reactions. We studied the therapeutic benefit vs risk of immune deviation in experimental allergic encephalomyelitis of SJL mice induced by MP4, a myelin basic protein-proteolipid protein (PLP) fusion protein. MP4 administration in IFA induced type 2 T cell immunity, IgG1 Abs, and experimental allergic encephalomyelitis protection, and all three were enhanced by repeat injections. Despite high Ab titers, anaphylactic side reactions were not observed when MP4 was repeatedly injected in IFA or as soluble Ag s.c. In contrast, lethal anaphylaxis was seen after s.c. injection of soluble PLP:139-151 peptide, but not when the peptide was reinjected in IFA. Therefore, the Ab response accompanying the immune therapy constituted an anaphylactic risk factor only when the autoantigen was not retained in an adjuvant and when it was small enough to be readily disseminated within the body. Taken together, our data show that treatment regimens can be designed to boost the protective type 2 T cell response while avoiding the risk of Ab-mediated allergic side effects.     

Prevention of anaphylactic death by macrophage blockade.

Data in the literature concerning the role of macrophages in anaphylaxis are contradictory. In the present study the effect of macrophage blockade induced by gadolinium chloride (GdCl3) on anaphylactic shock was investigated. Our observations show that GdCl3 prevents the lethal anaphylactic shock of mice sensitized to ovalbumin. GdCl3 given i.v. in a dose of 1 mg/100 g body weight 24 or 48 h before the elicitation of anaphylactic shock resulted in 90% survival, compared to the 43% survival in the control group. The same dose of this rare earth metal salt also greatly reduced the mortality in mice sensitized with ovalbumin containing Bordetella pertussis vaccine, and the symptoms of anaphylaxis including the accumulation of 5-hydroxytryptamine in the liver. Our results suggest that macrophages play an important role in anaphylaxis.     

Focus on the agents most frequently responsible for perioperative anaphylaxis

Adverse reactions (ARs) to drugs administered during general anesthesia may be very severe and life-threatening, with a mortality rate ranging from 3 to 9%. The adverse reactions to drugs may be IgE and non-IgE-mediated. Neuromuscular blocking agents (NMBA) represent the first cause of perioperative reactions during general anesthesia followed by latex, antibiotics, hypnotic agents, opioids, colloids, dyes and antiseptics (chlorhexidine). All these substances (i.e. NMBA, anesthetics, antibiotics, latex devices) may cause severe systemic non-IgE-mediated reactions or fatal anaphylactic events even in the absence of any evident risk factor in the patient’s anamnesis. For this reason, in order to minimize perioperative anaphylactic reactions, it is important to have rapid, specific, sensitive in vitro diagnostic tests able to confirm the clinical diagnosis of acute anaphylaxis.     

Incidence of anaphylaxis in the emergency department of a general hospital in Milan

Objective: To evaluate incidence and causes of anaphylactic reactions in the emergency room (E.R.) of a general hospital in Milan during a 2-year period. Methods: We retrospectively studied the computerized records of patients discharged from an E.R. with a diagnosis of anaphylactic reaction. Anaphylaxis was established on the presence of at least two cutaneous, respiratory, gastrointestinal or cardiovascular system symptoms. Results: During 1997 and 1998, out of 38 685 patients referred to the E.R., 13 had severe anaphylaxis with loss of consciousness (LOC) and 127 had anaphylactic symptoms, without LOC. Of the 13 patients with LOC, a possible cause was identified in 12 (five foods, six drugs, one hair dye). In the other 127 patients anaphylaxis was related to foods in 49 cases (38.5%), drugs in 44 (34.6%), unknown causes in 29 (22.8%), hymenoptera stings in two (1.5%), and other causes in three (2.3%). Conclusion: The incidence of anaphylactic reactions was 0.4% and mainly affected females and atopic subjects. Foods, particularly fruits and vegetables, appeared to be the most important cause; other important causes were non steroidal antiinflammatory drugs and β-lactam antibiotics.     

Phyllodes tumor of the breast. A cytohistologic study of 80 cases

OBJECTIVE: To study the cytologic features of phyllodes tumor (PT) of the breast and determine the accuracy of their subclassification in fine needle aspirates. STUDY DESIGN: Eighty cases of histologically diagnosed PT between 1982 and 1997 with a previous fine needle aspiration (FNA) were evaluated. The FNA smears of each case were reviewed without knowledge of the initial cytologic diagnosis and subclassified into benign, borderline or malignant PT. RESULTS: Benign PTs were characterized by a dimorphic mixture of stromal and epithelial cells. The stromal fragments showed mild to moderate cellularity with absent to minimal pleomorphism and no mitosis. There were occasional, if any, single stromal cells. Borderline PTs had stromal fragments with moderately cellular stroma exhibiting moderate pleomorphism. Two additional features were the presence of single stromal cells and an occasional mitosis in the stromal fragments/single cells. Aspirates from malignant PT were very cellular, with a high stromal/epithelial ratio and marked stromal cellularity. The stromal cells were highly pleomorphic, with frequent mitosis and atypical single stromal cells in the background. Fifty-seven of the 80 histologically documented cases (71.3%) were diagnosed as PT on FNA (40 benign, 10 borderline and 7 malignant). In 81% (46 of 57 PTs), good cytohistologic correlation (32 benign, 8 borderline and 6 malignant) was observed. In another eight cases, one grade differentiation between cytologic and histologic grade was observed. Six of the nine malignant PTs on histology were correctly subclassified on cytology. There were one false positive and two false negative cases. CONCLUSION: Cytologic diagnosis and grading of PT on FNA is possible. Special care should be undertaken in interpreting phyllodes fragments, cellularity of stroma, pleomorphism and mitosis. Single stromal cells are also important morphologic criteria for subclassification. Multiple-site aspiration is advisable to avoid diagnostic errors.     

Quantification (by nomograms) of physiopathological parameters in an experimental model of passive anaphylaxis caused by immune complexes

Pharmacological changes in a model of passive anaphylaxis by immune complexes in mice have been studied. The results have been evaluated by nomograms to overcome the difficulties observed when using classical criteria as 50% lethal dosis or 50% infective dosis in experimental models which do not allow serial dilutions of the reagents. Mean lethal time, maximal pathogenic capacity and relative survival time can be easily determined, allowing the comparison of the immunopharmacologic protection for each administration schedule. By these criteria, we have observed a different degree of anaphylactic reaction depending on the genetic background of the hosts as well as a protection against anaphylactic shock using the antiserotoninic cyproheptadine and, to a lesser extent, by the platelet antiaggregant ticlopidine.     

Contribution of classic and alternative effector pathways in peanut-induced anaphylactic responses

Food allergy affects approximately 5% of children and is the leading cause of hospitalization for anaphylactic reactions in westernized countries. However, the pathways of anaphylaxis in food allergy are still relatively unknown. We investigated the effector pathways of allergic and anaphylactic responses of different strains of mice in a clinical relevant model of peanut allergy. C3H/HeOuJ, C57BL/6 and BALB/c mice were sensitized by intragastric peanut extract and challenged by intragastric or intraperitoneal injection of peanut. Peanut-specific T cell responses, IgE, IgG1 and IgG2a and mucosal mast cell degranulation were induced to different extent in C3H/HeOuJ, C57BL/6 and BALB/c mice. Interestingly, anaphylactic symptoms after systemic challenge were highest in C3H/HeOuJ followed by C57BL/6 but were absent in BALB/c mice. Mechanistic studies showed that the food allergic systemic anaphylaxis was dependent on platelets, FcRγ and mast cells, and partially dependent on platelet activating factor and monocytes/macrophages, depending on mouse strain. These data demonstrate that in three mouse strains, components of the classic and alternative anaphylactic cascade are differently expressed, leading to differential outcomes in parameters of allergic disease and food induced systemic anaphylaxis.     

The spread and antibiotic resistance of Salmonella enterica serotype typhimurium DT104 in Hungary

Comparison of phage types (PTs) determined by Felix and Callow's and Anderson's methods was performed testing 99 human strains of S. enterica serotype Typhimurium (S. typhimurium) isolated in Hungary. PT2 and PT2c--according to Felix-Callow--corresponded with Anderson's DT104 in case of 39 strains out of 40. Among 59 isolates belonging to other Felix-Callow's PTs only one strain was found which was DT 104. Similar unambiguous equalities could not be established between any other PTs comparing the two methods. The PTs of 17,877 human strains isolated between 1988 and 1999 were determined using Felix-Callow's method. On the basis of the above equality the emergence of DT104 could be followed retrospectively by means of the rate of PT2 and PT2c. The increase of DT104 began already in 1989, emerging first PT2c then PT2. It predominated since 1991 and it reached its maximum (78.3%) in 1999. The incidence of multiresistance among one of the groups of DT104 strains (Felix-Callow's PT2) was significantly higher in 1998 than the average of non-DT104 strains. The predominant R-type was ACST.     

A biotinylated peptide,BP21, alleviates hypotension in anaphylactic mice

Platelet‐activating factor (PAF) is known as an important mediator of anaphylaxis and, therefore, may possibly serve as a direct target for anti‐anaphylactic drugs. We recently reported that a synthetic N‐terminally biotinylated peptide, BP21, alleviates hypothermia and vascular hyperpermeability during anaphylactic reactions in a mouse model of anaphylaxis via the direct binding of a Tyr‐Lys‐Asp‐Gly sequence in the peptide to PAF. In this study, we investigated the effect of BP21 on in vivo anaphylactic hypotension. Results showed that BP21 significantly inhibited anaphylactic hypotension in a dose‐dependent manner, with peak severity being reached within 20 minutes. Adrenaline, which is the recommended first line treatment for anaphylactic patients, did not inhibit anaphylactic hypothermia. The combined administration of BP21 with adrenaline inhibited both hypotension and hypothermia, even at both low doses, more effectively compared with solo administration of BP21 or adrenaline. These results suggested that BP21 could potentially be a novel anti‐anaphylactic agent for targeting PAF in vivo.     

Medium components adsorbed to mycoplasmal cells

Washed cells of Mycoplasma pneumoniae mixed with complete Freund's adjuvant were capable of producing lethal anaphylactic shock in mice when injected repeatedly into the foot pad. The causative agent is associated with horse serum contained in the culture medium but cannot be removed from mycoplasmal cells by repeated washing with phosphate buffered saline. Mycoplasmas grown in medium containing rabbit serum had a similar effect. No lethal anaphylaxis occurs when M. pneumoniae is injected without adjuvant.     

The diagnostic value of Western blot method in patients with cystic echinococcosis

Cystic echinococcosis (CE) is the larval cystic stage (called echinococcal cysts) of a small taeniid-type tapeworm (Echinococcus granulosus). Carnivores such as dogs are usually definitive hosts. Intermediate hosts are typically herbivores such as sheep and cattle. CE can be detected using various imaging techniques such as ultrasonography or radiology. Moreover the primary diagnosis has to be confirmed by serological tests since the clinical signs of the disease are non-specific. This study examined the antigenic band patterns useful for serologic diagnosis of hydatidosis. We also report on the post-operative evolution of patients treated for this disease and also determined the diagnostic performance of Western blot IgG kit. Twenty-five (16 females and 9 males) non-operated patients with hydatid cysts (NOP) and 33 (21 females and 12 males) operated patients with hydatid cysts (OP) were included as study group and 22 healthy individuals (14 females and 8 males) with no known chronic diseases were included as a control group. The ages of the patients and control group individuals were between 16-83 years. Patient and control groups were matched for age and sex. Cyst hydatid IgG antibodies were detected in the sera from all patient groups but no antibodies were found in the sera from the control group using ELISA IgG method. Twenty-three (92%) non-operated patients and 18 (54.5%) operated patients exhibited positive results when Western blot IgG kit was used. The P7 band pattern was detected in the sera from all operated and non-operated patients. Twenty-seven of these positive cases had p7 and (p7+p16/18), (p7+p24/26) or (p7+p16/18+p24/26). No antibodies against p7, p16/18 ve p24/26 band patterns were seen in sera from the control group A statistically significant difference was detected between operated and nonoperated patients for Western blot positivity.(p<0.01). p: 0.018- X2=5,604- OR: 0.176- 95% CI: 0.037- 0.841. The sensitivity, specificity, positive prediction and negative prediction values of Echinococcus granulosus Western blot kit for 25 cases with CE and 22 healthy controls were calculated as 92%, 100%, 100% and 91.7%, respectively. In conclusion, we suggest that monitoring p7 in all non-operated patients may be useful to determine the efficiacy of medical treatment and that monitoring p7 antibodies using serological and Western blot methods in operated patients may be useful for the screening of post-operative evolution in patients with hydatid cyst.     

Treatment of hepatic hydatid cysts by percutaneous aspiration and hypertonic saline injection: results of a cooperative work

Percutaneous puncture-aspiration-injection-reaspiration (PAIR) of hydatid liver cysts, was performed in 38 patients 14-80 years old, with a total of 60 liver hydatid cysts. After aspiration under computed tomography guidance, hypertonic saline was injected into the cystic cavities of patients as a scolecidal agent. No major complications were associated with the procedures. In the follow-up period of 18 months, control CT scans of 35 cysts revealed a gradual decrease in cyst size with a mean volume reduction of 66%. Complications included two cases of urticaria, one case of anaphylaxis and one subcapsular hematoma. No mortality occurred. It is concluded that percutaneous aspiration and hypertonic saline injection for liver hydatid cysts appears to be an effective form of treatment and may eventually prove to be an alternative to surgical intervention.     

Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model

Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods. Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions. We investigated the effects of s.c. injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy. PN-allergic C3H/HeJ mice were treated s.c. with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1-3 plus HKLM). The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined. Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1-3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5. Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced. This protective effect was markedly more potent than in the mAra h 1-3 protein alone-treated group. HKLM alone did not have any protective effect. Reduced IL-5 and IL-13, and increased IFN-gamma levels were observed only in splenocytes cultures from mAra h 1-3 plus HKLM-treated mice. These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA.     

Is caveolin involved in normal proximal tubule function? Presence in model PT systems but absence in situ

Kidney proximal tubule (PT) cells are specialized for the uptake and transport of ions, solutes, peptides, and proteins. These functions are often regulated by hormones that signal at the cell surface and are internalized by clathrin-mediated endocytosis. However, the caveolin/caveolae pathway has also been implicated in normal PT function, often based on data from isolated PTs or PT cells in culture. Although we reported previously that caveolae and caveolin 1 are not detectable in PTs in vivo, reports of caveolin expression and function in PT cells appear periodically in the literature. Therefore, we reexamined caveolin expression in PTs in vivo, in isolated "purified" PTs following collagenase digestion, and in cultured PT cells. Caveolin 1 and 2 protein, mRNA, or immunofluorescence was undetectable in PTs in vivo, but PT cell cultures expressed caveolin 1 and/or 2. Furthermore, caveolin 1 and 2 mRNAs were detected in isolated PTs along with the endothelial markers CD31 and ICAM1. In contrast, no caveolin or endothelial marker mRNAs were detectable in samples isolated from snap-frozen kidneys by laser cut microdissection, which eliminates contamination by other cell types. We conclude 1) caveolin 1 and 2 are not normally expressed by PT cells in situ, 2) caveolin expression is "activated" in cultured PT cells, 3) contamination with non-PT, caveolin-expressing cells is a potential source of caveolin 1 and 2 that must be taken into account when isolated PTs are used in studies to correlate expression of these proteins with PT function.     

Physico-chemical properties of microbial antigens and the immuno-allergic tests]

Experiments were conducted on guinea pigs sensitized by the delayed and immediate types of allergy. Different antigens obtained from the strain of Brucella abortus BA-19 were used for sensitization and the resolving action. Comparison of the resolving properties of the corpuscular, soluble (ultrasound treated) antigens and purified protein fractions, polysaccharide and RNA was carried out in the skin reactions of the immediate and delayed type, passive skin anaphylaxis, acute anaphylactic shock, and the Schults-Dale test. Immediate reactions to the purified protein fraction were weaker than those to the whole soluble antigen, by which the animals were sensitized. Polysaccharide and the RNA-fractions proved to be inactive in the allergic reactions.     

大连市两种来源的副溶血性弧菌分子分型

目的 分析食品来源、患者来源及2种来源的副溶血性弧菌之间的PFGE图谱的关系,从分子流行病学角度探讨2种来源的副溶血性弧菌的关联.方法 收集患者和食品2种来源的副溶血性弧菌178株,经限制性内切酶SfiI酶切,用脉冲场凝胶电泳方法进行电泳,凝胶成像仪获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析.结果 ...     

Antisense gene inhibition by phosphorothioate antisense oligonucleotide in Arabidopsis pollen tubes

Sexual reproduction is an essential biological event for proliferation of plants. The pollen tube (PT) that contained male gametes elongates and penetrates into the pistils for successful fertilization. However, the molecular mechanisms of plant fertilization remain largely unknown. Here, we report a transient inhibition of gene function using phosphorothioate antisense oligodeoxynucleotides (AS‐ODNs) without cytofectin, which is a simple way to study gene function in Arabidopsis thaliana PTs. The PTs treated with AS‐ODNs against both ANX1 and ANX2 showed short, knotted, and ruptured morphology in vitro/semi‐in vitro, whereas normal PT growth was shown in its sense control in vitro/semi‐in vitro. PT growth was impaired in a manner dependent on the dose of AS‐ODNs against both ANX1 and ANX2 above 10 μm . The treatment with AS‐ODNs against ROP1 and CalS5 resulted in waving PTs and in short PTs with a few callose plugs, respectively. The expression levels of the target genes in PTs treated with their AS‐ODNs were lower than or similar to those in the sense control, indicating that the inhibition was directly or indirectly related to the expression of each mRNA. The AS‐ODN against fluorescent protein (sGFP) led to reduced sGFP expression, suggesting that the AS‐ODN suppressed protein expression. This method will enable the identification of reproductively important genes in Arabidopsis PTs.     

Genetic and Clinical Characteristics of Phyllodes Tumors of the Breast

PURPOSE: Phyllodes tumors (PTs) of the breast are rare, accounting for less than 1% of all breast tumors. Among PTs, malignant PTs (MPTs) have malignant characteristics and distant metastases occur in about 20% to 30% of MPTs. However, there is no effective treatment for MPTs with distant metastasis, resulting in an abject prognosis. We performed targeted deep sequencing on PTs to identify the associations between genetic alterations and clinical prognosis. METHODS: We performed targeted deep sequencing to evaluate the genetic characteristics of PTs and analyzed the relationships between clinical and genetic characteristics. RESULTS: A total of 17 PTs were collected between 2001 and 2012. Histologic review was performed by pathologists. The samples included three benign PTs, one borderline PT, and 13 MPTs. The most frequently detected genetic alteration occurred in the TERT promoter region (70.6%), followed by MED12 (64.7%). EGFR amplification and TP53 alteration were detected in four MPTs without genetic alterations in MED12 and TERT promoter regions. Genetic alterations of RARA and ZNF703 were repeatedly found in PTs with local recurrence, and genetic alterations of SETD2, BRCA2, and TSC1 were detected in PTs with distant metastasis. Especially, MPT harboring PTEN and RB1 copy number deletion showed rapid disease progression. CONCLUSIONS: In this study, we provide genetic characterization and potential therapeutic target for this rare, potentially lethal disease. Further large-scale comprehensive genetic study and functional validation are warranted.