Cystic fibrosis modifier genes related to Pseudomonas aeruginosa infection.

Cystic fibrosis (CF) is one of the most common life-shortening genetic disorders, and the CF transmembrane conductance regulator (CFTR) is the major causal gene. However, a substantial clinical variability among patients with identical CFTR genotypes suggests the presence of modifier genes. We tested the effect of four genes involved in Pseudomonas aeruginosa infection. Analysis of a primary cohort detected eight candidate polymorphisms that were genotyped in the secondary cohort of 1579 patients; lung function and age at first infection with P. aeruginosa were considered as the phenotypes. Both additive and codominant models were considered, adjusting for confounding variables but not for multiple comparisons. In the secondary cohort, heme oxygenase-1 (HMOX1) rs2071749 had the most significant effect on lung function in the pediatric group (P=0.01; P(corrected)=0.03), and complement factor 3 (C3) rs11569393 and HMOX1 rs2071746 in the adult groups (P=0.03 for both variants; P(corrected)=0.16, 0.09). No polymorphism of complement factor B (CFB) or toll-like receptor 4 (TLR4) had a significant modifying effect on lung function in either group. We have identified two genes that showed nominal association with disease severity among CF patients. However, because of the multiple comparisons made, further studies are required to confirm the interaction between these modifying genes and CFTR.

Understanding the population structure of North American patients with cystic fibrosis.

It is generally presumed that the cystic fibrosis (CF) population is relatively homogeneous, and predominantly of European origin. The complex ethnic make-up observed in the CF patients collected by the North American CF Modifier Gene Consortium has brought this assumption into question, and suggested the potential for population substructure in the three CF study samples collected from North America. It is well appreciated that population substructure can result in spurious genetic associations. To understand the ethnic composition of the North American CF population, and to assess the need for population structure adjustment in genetic association studies with North American CF patients, genome-wide single-nucleotide polymorphisms on 3076 unrelated North American CF patients were used to perform population structure analyses. We compared self-reported ethnicity to genotype-inferred ancestry, and also examined whether geographic distribution and cystic fibrosis transmembrane regulator (CFTR) mutation type could explain the population structure observed. Although largely Caucasian, our analyses identified a considerable number of CF patients with admixed African-Caucasian, Mexican-Caucasian and Indian-Caucasian ancestries. Population substructure was present and comparable across the three studies of the consortium. Neither geographic distribution nor CFTR mutation type explained the population structure. Given the ethnic diversity of the North American CF population, it is essential to carefully detect, estimate and adjust for population substructure to guard against potential spurious findings in CF genetic association studies. Other Mendelian diseases that are presumed to predominantly affect single ethnic groups may also benefit from careful analysis of population structure.

Do common in silico tools predict the clinical consequences of amino-acid substitutions in the CFTR gene?

Computational methods are used to predict the molecular consequences of amino-acid substitutions on the basis of evolutionary conservation or protein structure, but their utility in clinical diagnosis or prediction of disease outcome has not been well validated. We evaluated three popular computer programs, namely, PANTHER, SIFT and PolyPhen, by comparing the predicted clinical outcomes for a group of known CFTR missense mutations against the diagnosis of cystic fibrosis (CF) and clinical manifestations in cohorts of subjects with CF-disease and CFTR-related disorders carrying these mutations. Owing to poor specificity, none of tools reliably distinguished between individual mutations that confer CF disease from mutations found in subjects with a CFTR-related disorder or no disease. Prediction scores for CFTR mutations derived from PANTHER showed a significant overall statistical correlation with the spectrum of disease severity associated with mutations in the CFTR gene. In contrast, PolyPhen- and SIFT-derived scores only showed significant differences between CF-causing and non-CF variants. Current computational methods are not recommended for establishing or excluding a CF diagnosis, notably as a newborn screening strategy or in patients with equivocal test results.

Plasmablastic lymphoma presenting in a human immunodeficiency virus-negative patient: a case report.

Plasmablastic lymphoma (PBL), an aggressive non-Hodgkin's lymphoma that carries a poor prognosis, previously has been identified almost exclusively in patients infected with the human immunodeficiency virus (HIV). We present a case of a 42-year-old HIV-negative patient presenting with an isolated nasal cavity mass, the typical presentation for PBL. The patient was given systemic chemotherapy, central nervous system prophylaxis, and consolidative locoregional radiotherapy and achieved a complete clinical response. This case suggests PBL should be considered in HIV-negative patients with characteristic findings.

Age variation in Hodgkin's disease risk factors in older women: evidence from a population-based case-control study.

Hodgkin's disease (HD), which affects all age groups, has been associated with childhood social class, particularly among adults under age 40. Little is known about social class risk factors in older adults, and the few existing studies have conflicting findings. As part of a population-based case-control study of HD in women, we examined social class risk factors by diagnostic age groups (45-54 years and 55-79 years) corresponding to incidence patterns and by histologic subtypes based on a uniform pathologic review. Among women ages 45-54, cases were more likely to be Catholic, to have lower income and to be taller than controls. Among women ages 55-79, cases tended to have come from small or large childhood households, lived in single-family childhood housing, and had a single rather than shared bedroom at age 11. For the nodular sclerosis (NS) histologic subtype, similar age differences in risk factors were apparent. Comparisons between the NS and non-NS subtypes in women ages 55-79 identified some common risk factors (single-family childhood home, single bedroom at age 11) but others specific to one subtype (childhood household size, adult height for NS; lower maternal education for non-NS). Thus, some social class associations with HD differed between middle-aged and older women, as well as between these groups and younger adults, while others were shared across age groups. Risk also was associated with both higher and lower childhood social class in middle-aged and older women, in contrast with previous findings. None of these patterns was explained entirely by histologic subtype but may reflect age and histology subtype variation in the HD-EBV association.

Expert review of the diagnosis and histologic classification of Hodgkin disease in a population-based cancer registry: interobserver reliability and impact on incidence and survival rates.

BACKGROUND: The reliability of Hodgkin disease (HD) diagnosis and histologic classification is an ongoing concern but has not been evaluated in a population-based case series in 20 years. Yet, diagnostic error in cancer registry data used in surveying HD occurrence may produce statistics that misrepresent incidence, mortality, or survival. METHODS: Uniform pathology review was attempted for all 395 women ages 19--79 years with incident HD reported to a population-based cancer registry in 1988--94. Agreement between original registry and review diagnoses was measured with positive predictive values and kappa statistics. Incidence rates and survival probabilities were computed based on registry and review diagnoses. RESULTS: Registry and review diagnosis agreed for 245 of the 362 reviewed cases. Positive predictive values varied by histologic subtype (nodular sclerosis, 95%; lymphocyte predominance, 69%; mixed cellularity, 58%; lymphocyte depletion, 0%; not otherwise specified, 40%), but agreement was good overall (kappa, 0.66, 95% confidence interval, 0.56--0.76). Eleven patients were determined not to have HD; all were older than age 44 years. Hodgkin disease incidence rates differed for original and review diagnoses only in older women, for whom registry rates slightly overestimated incidence. Five-year survival rates did not differ for registry and review data overall or by age group. CONCLUSIONS: For most adult women patients, the diagnosis of HD was confirmed on review, reflecting the very good agreement between registry and review diagnoses for nodular sclerosis, the most common subtype. Thus, cancer registry statistics for this time period can provide accurate estimates of disease patterns for HD overall and for the nodular sclerosis variant. For other histologic subtypes, rates may be unreliable, and HD occurrence overall may be less dependable in populations with larger proportions of these subtypes.

Epstein-Barr virus and survival after Hodgkin disease in a population-based series of women.

BACKGROUND: Epstein-Barr virus (EBV) positive Hodgkin disease (HD), as defined by the presence of EBV genes or gene products in the malignant cells, differs epidemiologically from EBV negative HD. However, survival patterns for EBV-defined HD have not been well studied. To determine if EBV status influenced survival time after HD, the authors investigated a large, population-based series of female patients. METHODS: For 311 female patients living in the Greater San Francisco Bay Area who were aged 19-79 years with HD diagnosed between mid-1988 and 1994, histopathologically rereviewed archived biopsy specimens were assayed for EBV with immunohistochemistry and in situ hybridization. The 53 subjects with EBV positive and the 258 with EBV negative HD were observed for vital status through 1998; overall survival was analyzed with Kaplan-Meier and Cox proportional hazards regression methods. RESULTS: Epstein-Barr virus positive HD patients were older, received diagnosis at a later stage, and were less likely to have nodular sclerosis histology than EBV negative patients. Deaths were reported for 21 (40%) EBV positive and 37 (14%) EBV negative patients. No survival differences were observed between EBV positive and negative women aged 19-44 years, but survival was significantly poorer in women aged 45-79 years with EBV positive HD. Regression analysis confirmed this strong negative effect of EBV positive status on survival (hazard ratio for death, 3.0; 95% confidence interval, 1.5-6.2) as unrelated to age, stage at diagnosis, or tumor histology. CONCLUSIONS: This study found a marked survival disadvantage for EBV positive HD in older but not young adult women. These findings suggest influences of both EBV status and age on HD survival, as well as pathogenesis.

Biphasic activation of the BMP pathway patterns the Drosophila embryonic dorsal region.

The BMP pathway patterns the dorsal region of the Drosophila embryo. Using an antibody recognizing phosphorylated Mad (pMad), we followed signaling directly. In wild-type embryos, a biphasic activation pattern is observed. At the cellular blastoderm stage high pMad levels are detected only in the dorsal-most cell rows that give rise to amnioserosa. This accumulation of pMad requires the ligand Screw (Scw), the Short gastrulation (Sog) protein, and cleavage of their complex by Tolloid (Tld). When the inhibitory activity of Sog is removed, Mad phosphorylation is expanded. In spite of the uniform expression of Scw, pMad expansion is restricted to the dorsal domain of the embryo where Dpp is expressed. This demonstrates that Mad phosphorylation requires simultaneous activation by Scw and Dpp. Indeed, the early pMad pattern is abolished when either the Scw receptor Saxophone (Sax), the Dpp receptor Thickveins (Tkv), or Dpp are removed. After germ band extension, a uniform accumulation of pMad is observed in the entire dorsal domain of the embryo, with a sharp border at the junction with the neuroectoderm. From this stage onward, activation by Scw is no longer required, and Dpp suffices to induce high levels of pMad. In these subsequent phases pMad accumulates normally in the presence of ectopic Sog, in contrast to the early phase, indicating that Sog is only capable of blocking activation by Scw and not by Dpp.

Noncovalent interactions of the Apple 4 domain that mediate coagulation factor XI homodimerization.

The Apple 4 (A4) domain of human plasma factor XI (FXI) was used to investigate the process of FXI noncovalent dimer formation. Recombinant 6-histidine-tagged A4 domain proteins were prepared utilizing a bacterial expression system. Purification was accomplished under denaturing conditions, followed by a refolding protocol to facilitate correct disulfide bond formation. Analysis of the A4 domain (C321S mutant) by size exclusion chromatography indicated the presence of a slowly equilibrating reversible monomer-dimer equilibrium. The elution profiles reveal highly symmetrical peaks for both dimeric and monomeric species with elution times that were highly reproducible for varying amounts of both the dimeric and monomeric species. The monomer-dimer equilibrium was found to be dependent upon changes in both pH and salt concentration. Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by a dissociation constant (K(D)) value of 229 +/- 26 nm with a calculated Delta G value of 9.1 kcal/mol. This report identifies electrostatic contributions and the presence of a hydrophobic component that mediate interactions at the A4 domain interface. The rate of dissociation for the recombinant A4 domain C321S mutant was examined by monitoring the increase in 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt fluorescence under dissociating conditions, giving a value for a dissociation rate constant (k(off)) of 4.3 x 10(-3) s(-1).

The digestive system manifestations of Rosai-Dorfman disease (sinus histiocytosis with massive lymphadenopathy): review of 11 cases.

Rosai-Dorfman disease (RDD), originally described as sinus histiocytosis with massive lymphadenopathy, is a rare histiocytic proliferative disorder with a distinctive microscopic appearance. Formerly thought to be a process limited to lymph nodes, involvement by RDD has now been documented in many organ systems, notably bone, skin and soft tissue, central nervous system, eye and orbit, and upper respiratory tract. The digestive system, however, is affected only exceptionally, as reflected by the existence of only a handful of individual case reports. In this article, we report 11 patients in which the disease involved intestinal tract, liver, or pancreas, and describe the most salient clinicopathologic features. The specific site of involvement within the digestive system was gastrointestinal tract in 5, liver in 5, and pancreas in 1. Most patients also had evidence of disease in other extranodal sites, as well as in 1 or more lymph node groups.

Aggressive cutaneous NK and NK-like T-cell lymphomas: clinicopathologic, immunohistochemical, and molecular analyses of 12 cases.

Natural killer (NK) and NK-like T-cell lymphomas are rare hematolymphoid malignancies that predominate in the upper aerodigestive system. They also involve other extranodal sites, including the skin. Primary cutaneous manifestations of NK and NK-like T-cell lymphomas are uncommon, and the clinicopathologic features are poorly understood. We have studied 12 patients of varied ethnic backgrounds with CD56-positive lymphomas in the skin. Six patients subsequently progressed to disseminated disease. These lymphomas showed the following immunophenotype: CD56+, CD43+, TCRb-, CD3-/+, CD20-, CD30-/+, CD4-, and CD8-. Two cases exhibited T-cell receptor gene rearrangements supporting a T-cell origin for these lymphomas, whereas the remaining 10 cases were likely derived from NK cells. Our results show inconsistent association of these lymphomas with Epstein-Barr virus (EBV), the multidrug resistance phenotype, and expression of P53. In addition, we found a previously unreported correlation between lymphomas harboring EBV mRNA and the expression of the multidrug resistance phenotype. These lymphomas were aggressive and were associated with rapid clinical progression, treatment failure, multiple relapses, and an average survival of 15 months from the time of diagnosis. Our results indicate the importance of recognizing this disease as a distinct subset of aggressive cutaneous lymphomas that may be diagnosed on the basis of morphology, immunophenotype, and gene rearrangement studies.

Breast involvement by extranodal Rosai-Dorfman disease: report of seven cases.

Seven cases of breast involvement by extranodal Rosai-Dorfman disease are presented. The patients were women and their ages ranged from 15 to 84 years. Three patients had disease confined to the breast; one had involvement of the breast and ipsilateral axillary lymph nodes, and two had bilateral breast involvement as well as disseminated systemic disease. In all cases the clinical and radiographic presentation of the breast lesion raised the possibility of a malignant tumor. All but one of the lesions were treated by excisional biopsy. Microscopically, the lesions were relatively circumscribed, often multinodular masses, located in the breast stroma, with or without associated involvement of the subcutaneous tissue and dermis. They were composed of sheets of S-100 protein-positive large histiocytes displaying lymphocytophagocytosis, scattered in a polymorphous background of mature lymphocytes and plasma cells. The microscopic differential diagnosis includes idiopathic granulomatous mastitis, infective granulomas, Langerhans' cell histiocytosis, Erdheim-Chester disease, fibrous histiocytoma, and malignant melanoma.

Effects of low-intensity AC and/or DC electromagnetic fields on cell attachment and induction of apoptosis.

Rat tendon fibroblast (RTF) and rat bone marrow (RBM) osteoprogenitor cells were cultured and exposed to AC and/or DC magnetic fields in a triaxial Helmholtz coil in an incubator for up to 13 days. The AC fields were at 60 and 1000 Hz and up to 0.25 mT peak to peak, and the DC fields were up to 0.25 mT. At various combinations of field strengths and frequencies, AC and/or DC fields resulted in extensive detachment of preattached cells and prevented the normal attachment of cells not previously attached to substrates. In addition, the fields resulted in altered cell morphologies. When RTF and RBM cells were removed from the fields after several days of exposure, they partially reattached and assumed more normal morphologies. An additional set of experiments described in the Appendix corroborates these findings and also shows that low-frequency EMF also initiates apoptosis, i.e., programmed cell death, at the onset of cell detachment. Taken together, these results suggest that the electromagnetic fields result in significant alterations in cell metabolism and cytoskeleton structure. Further work is required to determine the relative effect of the electric and magnetic fields on these phenomena. The research has implications for understanding the role of fields in affecting bone healing in fracture nonunions, in cell detachment in cancer metastasis, and in the effect of EMF on organisms generally.

Congenital toxoplasmosis transmitted from an immunologically competent mother infected before conception.

Congenital transmission of Toxoplasma gondii from a mother who was apparently immunologically competent and who had toxoplasmic lymphadenitis 2 months before conception is described. Since no T. gondii-specific serological data were available for this mother from the time her lymph node biopsy specimen was obtained, the specimen was studied by polymerase chain reaction (PCR) to determine whether the T. gondii B1 gene was present. The predictive diagnostic value of histologic findings previously considered to be classic signs of T. gondii lymphadenitis also was studied. This was done by correlation of serological tests diagnostic of acute acquired T. gondii infection and presence of characteristic findings in biopsy specimens from persons without known immunocompromise. Both PCR and review of the characteristic features of her lymph node biopsy specimen confirmed the diagnosis of preconceptual infection in the mother. We also discuss two other cases in which apparently immunologically competent mothers with preconceptually acquired infection transmitted this parasite to their fetuses.

Focal changes of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease) associated with nodular lymphocyte predominant Hodgkin's disease.

Sinus histiocytosis with massive lymphadenopathy (SHML/Rosai-Dorfman disease) has, on rare occasions been identified as an isolated phenomenon in lymph nodes affected by malignant lymphomas. The Registry includes four cases of SHML in patients with non-Hodgkin's lymphomas and one with multiple myeloma. SHML has more recently been recorded as a focal finding in lymph nodes involved by Hodgkin's disease of the mixed cellularity type. We report two patients presenting with lymphadenopathy caused by involvement by nodular lymphocyte predominant Hodgkin's disease with focal changes of SHML, an association not previously recorded in the literature. Responsiveness of the histiocytic cells of SHML to B-cell derived cytokines is postulated as a mechanism for this phenomenon, an hypothesis previously raised in regard to the association of focal Langerhans cell histiocytosis with Hodgkin's disease and with non-Hodgkin's lymphomas.

Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase.

Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S. typhimurium OPRTase. Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines. Together with information from X-ray crystallography [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754], sequence comparisons, and chemical modification [Grubmeyer, C., Segura, E., & Dorfman, R. (1993) J. Biol. Chem. 268, 20299-20304], this work permits the assignment of functions of the five conserved lysines. Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity. Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10-fold decreases in kcat. Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes. Mutation of Lys-73 produced a 50-100-fold decrease in kcat and an 8-12-fold increase in the KM value for PRPP. Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and function of Salmonella typhimurium orotate phosphoribosyltransferase: protein complementation reveals shared active sites.

A solvent-exposed loop, comprising residues 98-119 of S. typhimurium orotate phosphoribosyltransferase (OPRTase), is at the subunit interface of the dimeric enzyme, and its amino acid side chains potentially contact active sites on either subunit. A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme.OMP [Scapin, G., Grubmeyer, C., & Sacchettini, J. C. (1994) Biochemistry 33, 1287-1294] and enzyme.PRPP.orotate complexes [Scapin, G., Ozturk, D. H., Grubmeyer, C., & Sacchettini, J. C. (1995) Biochemistry 34, 10744-10754]. Lys-103, which is essential for activity [Ozturk, D. H., Dorfman, R. H. Scapin, G., Sacchettini, J. C., & Grubmeyer, C. (1995) Biochemistry 34, 10755-10763], may thus be functional in the active site formed by the adjacent subunit. Asp-125 is an essential residue that is in the middle of the active site. Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produced approximately 21-23% of the enzymatic activity of the wild-type OPRTase. Heterodimer formation in the complemented mixtures was evidenced by various physical methods. Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit. As predicted from the three-dimensional structure, increased activity resulting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtures of D125N and either K73A or K73Q mutants. Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme. A K103A/D125N double mutant enzyme was also constructed and was able to inactivate wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Screening for depression among a well elderly population.

This article describes a geriatric wellness program in which social work practitioners played a major role. The focus of this article is twofold: to examine the use of a telephone screening test for depression among a well elderly population and to compare the results of that screening with the clinical judgment of social workers. Overall findings indicated that a telephone screening instrument incorporating the Rand Mental Health Inventory and the Center for Epidemiological Studies Depression Scale was an efficient tool for assessing a population with a higher rate of major depression. Furthermore, the social workers identified many previously undetected cases of major depression, and a majority of people referred for treatment completed those referrals.

Detection of immunophenotypic abnormalities in paraffin-embedded B-lineage non-Hodgkin's lymphomas.

The authors analyzed the frequency of immunophenotypic abnormalities in 1,474 cases of routinely fixed, paraffin-embedded B-lineage non-Hodgkin's lymphomas. B-lineage was determined by immunoreactivity for CD20 (L26, 92%); CD45RA (4KB5, an additional 3%) or immunoglobulin (Ig) light chain restriction (remaining 5%). CD45RA was found to be especially helpful on Bouin's-fixed or decalcified tissue and Ig staining was most helpful in plasmacytoid lesions. Coexpression of the T-cell marker CD43 (Leu-22) was the most common immunophenotypic abnormality, seen in 60% of mantle cell lymphomas (MCL), 39% of CLL/small lymphocytic lymphomas, 16% of diffuse large cell lymphomas (DLCL), but only 5% of follicular lymphomas (FL). Antibodies to CD45RO (A6 and UCHL1) and CD3 (polyclonal) were useful in distinguishing infiltrating T cells from B cells coexpressing CD43. Ig light chain restriction was the next commonest immunophenotypic abnormality, which was identified in 67% of plasmacytoid diffuse small cell lymphomas, 43% of MCLs, 35% of monocytoid B-cell lymphomas and 28% of FLs. Overexpression of bcl-2 oncogenic protein was observed in 71% of FLs (n = 96), but not in a control group of reactive follicular hyperplasias (n = 34). Combining two criteria increased the sensitivity of immunodiagnosis in certain circumstances.