Identification of alpha-gal sensitivity in patients with a diagnosis of idiopathic anaphylaxis.

IgE antibodies (Ab) specific to galactose-α-1,3-galactose (alpha-gal) are responsible for a delayed form of anaphylaxis that occurs 3-6 hours after red meat ingestion. In a unique prospective study of seventy participants referred with a diagnosis of idiopathic anaphylaxis (IA), six (9%) were found to have IgE to alpha-gal. Upon institution of a diet free of red meat, all patients had no further episodes of anaphylaxis. Two of these individuals had indolent systemic mastocytosis (ISM). Those with ISM had more severe clinical reactions but lower specific IgE to alpha-gal and higher serum tryptase levels, reflective of the mast cell burden. The identification of alpha-gal syndrome in patients with IA supports the need for routine screening for this sensitivity as a cause of anaphylaxis, where reactions to alpha-gal are delayed and thus may be overlooked.

Development and validation of the mastocytosis quality of life questionnaire: MC-QoL.

BACKGROUND: Mastocytosis is a heterogeneous disease characterized by a clonal expansion of mast cells in various organs. The vast majority of patients affected suffer from signs and symptoms caused by mediator release from mast cells. Although the disease burden is high, there is currently no specific instrument to measure health-related quality of life (HRQoL) impairment in patients with mastocytosis. OBJECTIVE: The aim of this study was to develop and validate a disease-specific tool to assess HRQoL impairment in patients with cutaneous and indolent systemic mastocytosis, the Mastocytosis Quality of Life Questionnaire (MC-QoL). METHODS: Sixty-two potential MC-QoL items were developed in a combined approach consisting of semi-structured patient interviews, expert input and literature research. Item selection was performed by impact analysis with 76 patients and a final review for face validity. The resulting MC-QoL was tested for validity, reliability and influence factors. In parallel, an US American-English version of the MC-QoL was developed. RESULTS: A total of 158 patients (41 CM, 41 MIS and 76 ISM) took part in the MC-QoL validation study. The final 27-item questionnaire was found to have a four-domain structure ('symptoms', 'emotions', 'social life/functioning' and 'skin'), a valid total score and an excellent test-retest reliability. Multiple regression analysis revealed disease duration, but not age, gender or skin involvement to be a significant determinant of HRQoL impairment in mastocytosis. CONCLUSIONS: The MC-QoL is the first disease-specific HRQoL questionnaire for adult patients with cutaneous and indolent systemic mastocytosis. This short, validated and reliable instrument will serve as a valuable tool in future clinical studies and in routine patient care.

Management of colorectal cancer in patients with inflammatory bowel disease.

BACKGROUND: This study evaluated the clinicopathological features and survival rates of patients with inflammatory bowel disease who developed colorectal cancer (CRC). METHODS: A retrospective review was performed on a prospectively maintained institutional database (1981-2011) to identify patients with inflammatory bowel disease who developed CRC. Clinicopathological parameters, management and outcomes were analysed. RESULTS: A total of 2,843 patients with inflammatory bowel disease were identified. One thousand six hundred and forty-two had ulcerative colitis (UC) and 1,201 had Crohn's disease (CD). Following exclusion criteria, there were 29 patients with biopsy-proven colorectal carcinoma, 22 of whom had UC and 7 had CD. Twenty-six patients had a preoperative diagnosis of malignancy/dysplasia; 16 of these were diagnosed at surveillance endoscopy. Nodal/distant metastasis was identified at presentation in 47 and 71 % of the UC and CD group, respectively. Operative morbidity for UC and CD was 33 and 17 %, respectively. Despite the less favourable operative outcomes following surgery management of UC-related CRC, overall 5-year survival was significantly better in the UC group compared to the CD group (41 vs. 29 %; p = 0.04) reflecting the difference in stage at presentation between the two groups. CONCLUSIONS: Patients who undergo surgery for UC-related CRC have less favourable short-term outcomes but present at a less advanced stage and have a more favourable long-term prognosis than similar patients with CRC and CD.

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

Specific and nonspecific obstructive lung disease in childhood: causes of changes in the prevalence of asthma.

Reversible airway obstruction in childhood includes two major groups of patients: those with recurrent wheezing following bronchiolitis in early childhood, and those with allergic asthma, which represents an increasingly large proportion of cases through the school years. Over the last 40 years of the 20th century, allergic asthma has increased in many countries and in relation to several different allergens. Although this increase has differed in magnitude in different countries and also in the social groups most affected, it has had several features in common. The increase generally started between 1960 and 1970, has been progressive since then, and has continued into the 1990s without a defined peak. Among children 5-18 years of age, the increase has predominantly been among allergic individuals. Theories about the causes of the increase in asthma have focused on two scenarios: a) that changes in houses combined with increased time spent indoors have increased exposure to relevant allergens, or b) that changes in diet, antibiotic use, immunizations, and the pattern of infections in childhood have led to a change in immune responsiveness such that a larger section of the population makes T(H)2, rather than T(H)1 responses including IgE antibodies to inhalant allergens. There are, however, problems with each of these theories and, in particular, none of the proposed changes can explain the progressive nature of the increase over 40 years. The fact that the change in asthma has much in common with epidemic increase in diseases such as Type II diabetes or obesity suggests that similar factors could be involved. Several lines of evidence are reviewed that suggest that the decline in physical activity of children, particularly those living in poverty in the United States, could have contributed to the rise in asthma. The hypothesis would be that the progressive loss of a lung-specific protective effect against wheezing has allowed allergic children to develop symptomatic asthma. What is clear is that current theories do not provide either an adequate explanation of the increase or a practical approach to reversing the current trend.

Variation in IgG1 heavy chain allotype does not contribute to differences in biological activity of two human anti-Rhesus (D) monoclonal antibodies.

BACKGROUND: Pooled human anti-Rhesus D antiserum is currently administered for the prevention of RhD alloimmunization. Increased demand, and decreased supply, of donated pooled antiserum has led to the investigation of the suitability of human monoclonal anti-RhD antibodies for use in its place. However, it is unclear which biological properties of monoclonal antibodies are important for function in RhD-positive foetal red cell clearance and the prevention of alloimmunization. Various antibodies behave differently in a number of in vitro assays of biological function. OBJECTIVES: To compare the function and structure of two human anti-RhD IgG1 monoclonal antibodies which differ in their ability to promote red cell lysis in vitro. In particular to examine whether the functional differences correlate to differences in the IgG1 heavy chain constant region (allotype). STUDY DESIGN: We report here the cloning, characterization and re-expression in stable myeloma cell transformants of cDNAs coding for two such antibodies, secreted by the heterohybridoma cell lines ESD-1 (THERAD 03) and LHM 70/45.3 (THERAD 06). The cDNAs were then recombined to exchange portions of the Fc encoding regions and the recombinant antibodies were assayed in vitro to determine RhD-positive red cell-dependent activity. RESULTS: Recombinant THERAD 03 and 06 antibodies behaved identically to the parent antibodies. The 'inactive' THERAD 06 did not have biological activity reconstituted by exchange with the THERAD 03 Fc regions, nor was THERAD 03 activity abolished by the reciprocal Fc region exchange. CONCLUSIONS: Human monoclonal anti-RhD antibodies can be cloned and re-expressed in stable cell lines, and exhibit identical properties to the parent antibodies. Differences in biological activity cannot be attributed to differences in IgG1 heavy chain allotype.

Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth.

Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function.

Transforming growth factor beta 1 (TGF beta 1) reduces cellular levels of p34cdc2, and this effect is abrogated by adenovirus independently of the E1A-associated pRB binding activity.

We have used E1A probes to study the roles of the p34cdc2 kinase and the retinoblastoma tumor susceptibility gene product (pRB) in transforming growth factor beta 1 (TGF beta 1)-mediated growth suppression in mink lung epithelial (Mv1Lu) cells. In agreement with previous reports, we see a decline in p34cdc2 kinase activity and a loss of pRB phosphorylation after TGF beta 1 treatment. We report here that TGF beta 1 induces not only a change in p34cdc2 kinase activity but a strong repression of p34cdc2 synthesis. Loss of p34cdc2 kinase activity is not seen until the steady-state level of p34cdc2 declines, suggesting that the intra-cellular signals induced by TGF beta 1 affect p34cdc2 at the level of expression, rather than by altering the posttranslational modifications of p34cdc2 that regulate its kinase activity. Infection with adenovirus expressing either wild-type E1A or a mutant E1A (pm928) defective for pRB binding alleviated TGF beta 1-mediated suppression of DNA synthesis, indicating that E1A does not need to bind pRB physically to keep cell growth-suppressing functions from being activated by TGF beta 1. The E1A.928 mutant virus is able to maintain p34cdc2 expression and kinase activity, as well as pRB phosphorylation in the presence of TGF beta 1, which may account for its ability to maintain cell cycle activity without directly sequestering pRB. Overall our results suggest that TGF beta 1 acts by signaling changes at the level of control of G1 gene expression, not at the level of posttranslational modification of p34cdc2 or its substrates.

The survivors of childhood solid tumors.

With the improvement in cancer therapy in recent years, the number of cancer survivors is rapidly increasing. Potential late medical and psychosocial sequelae of cancer therapy are reviewed. A practical guide for the primary health care giver is provided.

Simian virus 40 large-T antigen expresses a biological activity complementary to the p300-associated transforming function of the adenovirus E1A gene products.

In this report we present evidence that simian virus 40 T antigen encodes a biological activity that is functionally equivalent to the transforming activity lost by deletion of the E1A p300-binding region. T-antigen constructs from which the pRb-binding region has been deleted are virtually unable to induce foci of transformed cells in a ras cooperation assay in primary baby rat kidney cells. Nevertheless, such a construct can cooperate with an E1A N-terminal deletion mutant, itself devoid of transforming activity, to induce foci in this assay. The heterologous trans-cooperating activity observed between E1A and T-antigen deletion products is as efficient as trans cooperation between mutants expressing individual E1A domains. The cooperating function can be impaired by a deletion near the N terminus of T antigen. Such a deletion impairs neither the p53-binding function nor the activity of the pRb-binding region.

Instability of succinyl ester linkages in O2'-monosuccinyl cyclic AMP-protein conjugates at neutral pH.

Chromatographic and immunological evidence is presented regarding the hydrolysis of the ester linkage of O2'-monosuccinyl cyclic AMP in neutral solutions. Such hydrolysis occurs whether the nucleotide derivative is present in free form in solution or conjugated through its succinyl carboxyl group via an amide bond to proteins. The latter process apparently occurs when succinyl cyclic AMP is conjugated to human serum albumin for use as an immunogen in the production of anti-cyclic AMP antibodies and when the derivative is coupled to the enzyme glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). The enzyme conjugate has been used in developing a homogeneous enzyme immunoassay for cyclic AMP. Inhibition of the catalytic activity of enzyme-cyclic AMP conjugates by anti-cyclic AMP antibody decreases with time, apparently due to the loss of cyclic AMP from enzyme-cyclic AMP conjugates stored in neutral solutions. In addition, the ability of free cyclic AMP to completely reverse the inhibition process decreases with time because of the presence of antibodies in the anti-cyclic AMP sera that apparently inhibit enzyme activity because of their binding specificity for the residual succinate-protein determinant sites of the enzyme conjugates. Lyophilization of the conjugates immediately after preparation helps to overcome the problem; however, in vivo hydrolysis of immunogens prepared with the succinyl cyclic AMP derivative may always occur. The consequence of this hydrolysis reaction and the subsequent formation of anti-succinyl-protein antibodies will be discussed with regard to existing RIAs for cyclic AMP and a new homogeneous enzyme immunoassay for the nucleotide.