Characterization of a novel breast carcinoma xenograft and cell line derived from a BRCA1 germ-line mutation carrier

A human tumor xenograft (L56Br-X1) was established from a breast cancer axillary lymph node metastasis of a 53-year-old woman with a BRCA1 germ-line nonsense mutation (1806C>T; Q563X), and a cell line (L56Br-C1) was subsequently derived from the xenograft. The xenograft carries only the mutant BRCA1 allele and expresses mutant BRCA1 mRNA but no BRCA1 protein as determined by immunoprecipitation or Western blotting. The primary tumor, lymph node metastasis, and xenograft were hypodiploid by DNA flow cytometry, whereas the cell line displayed an aneuploidy apparently developed via polyploidization. Cytogenetic analysis, spectral karyotyping, and comparative genomic hybridization of the cell line revealed a highly complex karyotype with numerous unbalanced translocations. The xenograft and cell line had retained a somatic TP53 missense mutation (S215I) originating from the primary tumors, as well as a lack of immunohistochemically detectable expression of steroid hormone receptors, epidermal growth factor receptor, human epidermal growth factor receptor 2 (HER-2), and keratin 8. Global gene expression analysis by cDNA microarrays supported a correlation between the expression profiles of the primary tumor, lymph node metastasis, xenograft, and cell line. We conclude that L56Br-X1 and L56Br-C1 are useful model systems for studies of the pathogenesis and new therapeutic modalities of BRCA1-induced human breast cancer.     

IFN-gamma-enhanced allergen penetration across respiratory epithelium augments allergic inflammation

BACKGROUND: Respiratory allergen contact is the critical event in the elicitation and boosting of allergen-specific immune responses, as well as in the induction of immediate and late inflammatory reactions. OBJECTIVE: We sought to investigate the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of intact respiratory epithelium and used purified iodine 125-labeled recombinant major birch pollen allergen (rBet v 1) to study the extent, kinetics, and factors influencing transepithelial allergen penetration. RESULTS: Culture supernatants from activated allergen-specific T H 1 clones decreased transepithelial resistance. A screening of various factors (histamine, IFN-gamma, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-8, IL-12, and TNF-alpha) identified IFN-gamma as a potent factor capable of reducing epithelial barrier properties and enhancing transepithelial allergen penetration. Increased submucosal allergen concentrations caused by IFN-gamma-mediated reduction of epithelial barrier function provoked a more than 7-fold augmentation of histamine release from sensitized basophils. CONCLUSION: These results demonstrate that the T H 1 cell-derived cytokine IFN-gamma facilitates allergen penetration through the respiratory epithelium and thereby can aggravate allergic inflammation.     

Chromosome analysis of 20 breast carcinomas: Cytogenetic multiclonality and karyotypic-pathologic correlations

Short-term cultures from 20 breast carcinomas were analyzed cytogenetically. A normal female chromosome complement was found in 4 cases. Clonal chromosome aberrations were detected in 16 tumors. In 10 tumors, multiple cytogenetic clones were found; in 2 cancers the clones were related, reflecting clonal evolution, but in the remaining 8 tumors the clones were cytogenetically unrelated, indicating clonal heterogeneity in the origin of the tumor parenchyma. Correlation analysis between karyotypic and pathologic parameters indicated that cases with complex karyotypes and/or cytogenetically unrelated clones, when compared with cases with a single simple karyotypic abnormality, were generally of higher histologic malignancy grade, had more mitoses in the histologic sections, and also more often had carcinoma in situ lesions in the same breast. © 1993 Wiley-Liss, Inc.     

MICA4/HLA-DRB1*04/TNF1 haplotype is associated with mixed connective tissue disease in Swedish patients

In order to investigate major histocompatibility complex (MHC) class I chain-related gene A (MICA), tumor necrosis factor (TNFa), -308TNFA, and human leukocyte antigen (HLA-DR/DQ) polymorphisms in mixed connective tissue disease (MCTD), we analyzed 24 patients and 229 healthy controls from Sweden. MICA and TNFa typing was performed by polymerase chain reaction (PCR) and genotyping. HLA-DR and -DQ were genotyped using PCR-sequence specific primers (PCR-SSP) and PCR-sequence-specific oligonucleotide probe (PCR-SSOP), respectively. For analysis of -308TNFA polymorphisms we performed PCR with restriction endonuclease enzymes. We found that the MICA5.1-5.1 genotype was positively associated with MCTD. Shared epitope genes (DRB1*01 and DRB1*04) were also significantly positively associated with MCTD. Polymorphism of -308TNFA was not differently distributed in MCTD patients compared with controls. Furthermore, we demonstrated that frequencies of three estimated haplotypes were increased in MCTD patients compared with controls. Interestingly, the haplotype with MICA allele 4 together with DRB1*04 and TNF1 alleles gives the most specific pattern for MCTD patients compared with controls. Our study demonstrates a clear contribution of HLA loci in susceptibility to MCTD in the Swedish population. Susceptibility to MCTD may be linked to the MICA4/HLA-DRB1*04/TNF1 haplotype and MICA 5.1-5.1 genotype. Mixed connective tissue disease was also associated with shared epitope genes, which in RA has been associated with a more severe disease. Whether these genotypes affect the clinical phenotype of MCTD needs to be determined.     

Dynamics of the Murine Humoral Immune Response to Neisseria meningitidis Group B Capsular Polysaccharide

Immunization with Neisseria meningitidis group B capsular polysaccharide (CpsB) elicited responses in adult mice that showed the typical dynamic characteristics of the response to a thymus-independent antigen, in contrast to the thymus-dependent behavior of antibody responses to CpsC. The former had a short latent period and showed a rapid increase in serum antibodies that peaked at day 5, and immunoglobulin M (IgM) was the major isotype even though IgG (mainly IgG2a and IgG2b) was also detectable. This response was of short duration, and the specific antibodies were rapidly cleared from the circulation. The secondary responses were similar in magnitude, kinetics, IgM predominance, and IgG distribution. Nevertheless, a threefold IgG increase, a correlation between IgM and IgG levels, and dose-dependent secondary responses were observed. Hyperimmunization considerably reinforced these responses: 10-fold for IgM and 300-fold for IgG. This favored isotype switch was accompanied by a progressive change in the subclass distribution to IgG3 (62%) and IgG1 (28%), along with the possible generation of B-cell memory. The results indicate that CpsB is being strictly thymus independent and suggest that unresponsiveness to purified CpsB is due to tolerance.

The capsular polysaccharide (Cps) of Neisseria meningitidis group B (CpsB), the major cause of meningococcal disease in developed countries (38), is a linear homopolymer of α(2→8)-linked sialic acid on host sialogangliosides and sialoproteins (12, 16) causes immunological tolerance to sequential CpsB epitopes, with the anti-CpsB antibodies being mainly, if not solely, directed against conformational determinants preferably expressed by chains of eight or more residues (10). The conformational antigenic nature and metastable spatial structure of CpsB (10, 19), in combination with its neuraminidase sensitivity, tendency to internal lactonization, and intramolecular self-cleavage under mild acidic conditions (22, 29), were proposed to explain its poor immunogenicity (35). According to this hypothesis, the interaction of CpsB with B cells is transitory and therefore unable to elicit an antibody response (34). Alternatively, the high expression of longer sialic acid polymers (>12 residues), having the same α(2→8) linkage in polysialylated glycoproteins of vertebrate fetal tissues as well as limited areas of the adult neural system (21, 42), has been proposed to induce tolerance also to the conformational epitopes of CpsB (11). A feasible mechanism for inducing and maintaining tolerance, however, is not known. In any event, the poor immunogenicity of CpsB is associated with the α(2→8) linkage. Purified CpsC, a homopolymer of α(2→9)-linked sialic acid, has been shown to be immunogenic in mice (48).Bacterial Cps complexed to protein carriers induces long-lasting immunoglobulin G (IgG) antibody responses in young children and mice, which is indicative of the Cps conversion to a T-cell-dependent (TD) antigen (18). In contrast, CpsB conjugated to tetanus toxoid (3, 8, 20) or complexed with meningococcal outer membrane proteins (OMPs) (23, 24) is able to induce only low levels of CpsB-specific IgM. In these responses, however, CpsB-specific IgG was detectable (3, 8, 23). Since in simple terms protection from these infectious agents is due to the presence of circulating specific antibodies (13) and bearing in mind that an artificial IgG immune response may initiate an autoimmune process (11), we studied the evolution over time of the serum antibodies and changes in isotype distribution obtained by immunization with the native form of CpsB—namely, live N. meningitidis—in order to further explore the underlying mechanisms in the generation of the immune responses to this peculiar autoantigen which has both epitopes disseminated in the host and epitopes of ontogenetic and topologically restricted expression, a situation reproduced in the mouse model.     

Three new families with arterial tortuosity syndrome

Arterial tortuosity syndrome (ATS) is a rare condition with autosomal recessive inheritance characterized by connective tissue abnormalities. The most specific clinical findings are cardiovascular anomalies including tortuosity, lengthening, aneurysm, and stenosis formation of major arteries. Also ventricular hypertrophy is frequently present. Other anomalies are skin hyperextensibility and cutis laxa, joint laxity or contractures of the joints, and inguinal herniae. Histology shows disruption of elastic fibers of the media. These features suggest that ATS is a connective tissue disorder. A biochemical or molecular defect has not yet been identified. We describe here nine additional ATS patients from three consanguineous Moroccan families and review a total of 35 patients with this uncommon condition.     

Cloning, Expression, and Sequencing of a Cell Surface Antigen Containing a Leucine-Rich Repeat Motif from Bacteroides forsythus ATCC 43037

Bacteroides forsythus is a recently recognized human periodontopathogen associated with advanced, as well as recurrent, periodontitis. However, very little is known about the mechanism of pathogenesis of this organism. The present study was undertaken to identify the surface molecules of this bacterium that may play roles in its adherence to oral tissues or triggering of a host immune response(s). The gene (bspA) encoding a cell surface-associated protein of B. forsythus with an apparent molecular mass of 98 kDa was isolated by immunoscreening of a B. forsythus gene library constructed in a lambda ZAP II vector. The encoded 98-kDa protein (BspA) contains 14 complete repeats of 23 amino acid residues that show partial homology to leucine-rich repeat motifs. A recombinant protein containing the repeat region was expressed in Escherichia coli, purified, and utilized for antibody production, as well as in vitro binding studies. The purified recombinant protein bound strongly to fibronectin and fibrinogen in a dose-dependent manner and further inhibited the binding of B. forsythus cells to these extracellular matrix (ECM) components. In addition, adult patients with B. forsythus-associated periodontitis expressed specific antibodies against the BspA protein. We report here the cloning and expression of an immunogenic cell surface-associated protein (BspA) of B. forsythus and speculate that it mediates the binding of bacteria to ECM components and clotting factors (fibronectin and fibrinogen, respectively), which may be important in the colonization of the oral cavity by this bacterium and is also a target for the host immune response.     

(ATT) trinucleotide repeats in the antithrombin gene and their use in determining the origin of repeated mutations

Two (ATT) trinucleotide repeat polymorphisms have been identified in the tails of Alu repeat elements in intron 5 of the antithrombin gene. The frequency and distribution of allele sizes for the Alu 5 and Alu 8 tail polymorphisms have been defined in a sample Caucasian population. The Alu 5 polymorphism has two alleles while that of Alu 8 has 10 alleles with a heterozygosity of 0.83. These polymorphisms have been used in combination with four previously described polymorphisms within the antithrombin gene to construct antithrombin gene haplotypes in the sample Caucasian population. Twenty-two different haplotypes were observed, with the Alu 8 polymorphism being particularly useful in subdividing the core haplotype based on the previously identified polymorphisms. The haplotype data were used to investigate the origin of repeat mutations within the antithrombin locus. We compared the haplotypes associated the mutant antithrombin genes in five families with the mutation 2759C→T (L99F) and five families with the mutation 5381C→T (R129Stop). The mutation 2759C→T (L99F), which occurs within a non-CpG dinucleotide, was carried on a gene associated with an identical haplotype in each of the five families. The mutation 5381C→T (R129Stop), a single base substitution within a CpG dinucleotide, was associated with at least two different haplotypes. The findings suggest a founder effect in the five families sharing the 2759C→T (L99F) and at least two independent origins for the CpG dinucleotide mutation 5381C→T (Rl29Stop). © 1994 Wiley-Liss, Inc.     

The developmentally regulated neural crest-associated glycotope HNK-1 predicts metastasis in cutaneous malignant melanoma

Aberrant glycosylation is a common feature of metastatic sub-clones of malignant tumours and in uveal melanoma in particular, the HNK-1 glycotope has been positively correlated with poor prognosis. So far, no such correlation has been investigated in cutaneous melanoma. In order to do so, HNK-1 expression was evaluated immunohistochemically in 100 primary cutaneous melanomas and correlated with metastasis after up to 10-years' follow-up. Furthermore, HNK-1 expression was analysed in metastatic deposits (19 distant cutaneous metastases and six sentinel lymph node metastases), as well as in benign nevi. Kaplan-Meier analysis revealed a positive association between HNK-1 expression and metastasis (p < 0.005) and multivariate Cox regression analysis adjusted for the standard prognostic markers ulceration and vertical tumour thickness confirmed HNK-1 expression as an independent prognostic marker. HNK-1 expression was preserved in 42% of the distant cutaneous metastases, but metastatic cells in lymph nodes were devoid of HNK-1 immunoreactivity. None of the benign pigmented lesions exhibited HNK-1 immunoreactivity. Expression of the HNK-1 glycotope in cutaneous malignant melanoma is an independent prognostic marker of metastasis. Differential HNK-1 expression at the metastatic sites implies that its expression is modulated by the surrounding environment. As HNK-1 is also transiently expressed during migration of melanocyte precursor cells derived from the neural crest, recapitulation of this transient expression might occur during metastatic spread of cutaneous malignant melanoma.     

Die Bedeutung des Natriums für den renal-tubulären Calcium-Transport bei Rana ridibunda

Zusammenfassung In Anlehnung an frühere Experimente zur Na⁺-Abhängigkeit der renal-tubulären Transporte von Glucose, PAH und Urea wurden Versuche über die Bedeutung des Na⁺ für die Ca⁺⁺-Reabsorption an der isolierten künstlich perfundierten Niere von Rana ridibunda durchgeführt.Erniedrigung des Na⁺-Angebotes von 76,5 auf 20,0 mMol/l vermindert sowohl die Menge reabsorbierten Natriums als auch die des reabsorbierten Calciums, letztere allerdings weniger als erstere. Der Quotient reabsorbiertes Na⁺/reabsorbiertes Ca⁺⁺ verschiebt sich zugunsten des Ca⁺⁺.Fehlendes Na⁺-Angebot auf der Lumen- oder der Blutseite der Tubulusepithelien hat keinen sicheren Einfluß auf die Menge reabsorbierten Calciums, dagegen sinkt die Reabsorptionsrate von Ca⁺⁺ auf weniger als 10% des filtrierten, wenn Na⁺ weder auf der Lumen- noch auf der Blutseite angeboten wird.Die Ca⁺⁺-Reabsorptionsrate ist gesteigert bei Angebot von Ca⁺⁺ ausschließlich auf der Lumenseite, vermutlich wegen des unter physiologischen Bedingungen nicht existenten Konzentrationsgradienten, der blutwärts gerichtet ist. Bei Angebot von Ca⁺⁺ nur von der Blutseite erscheint kein Ca⁺⁺ im Harn: die Tubulusepithelien sind für Ca⁺⁺ im Richtungssinne der Sekretion undurchlässig.Durch die Hemmstoffe der Na⁺-Reabsorption Convallatoxin und Furosemid wird auch die Reabsorption von Ca⁺⁺ — sogar relativ stärker als die von Na⁺ — gehemmt. Da in den Versuchen mit Hemmstoffen das Na⁺-Angebot (extracelluläre Konzentration) konstant bleibt, wird geschlossen, daß die Ca⁺⁺-Reabsorption mit dem Na⁺ über dessen Transport gekoppelt ist.Im Prinzip folgt die Verknüpfung der Ca⁺⁺-Reabsorption mit dem Na⁺ den gleichen Gesetzmäßigkeiten wie die Verknüpfung des Na⁺ mit den Transporten von Glucose, PAH und Harnstoff. Damit ist ein weiteres Indiz für die Vermutung gewonnen, daß die Na⁺-Abhängigkeit ein zellphysiologisches Prinzip von weiter Gültigkeit ist.

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Summary With regard to earlier experiments about the influence of Na⁺ on the tubular transport of glucose, PAH and urea, the effect of Na⁺ on the reabsorption of Ca⁺⁺ in the isolated artificially perfused kidney of Rana ridibunda was studied.Lowering of the Na⁺-concentration from 76.5–20.0 mMol/l results in a decrease of the reabsorbed amounts of both Na⁺ and Ca⁺⁺, the decrease being less marked in the latter. The quotient Na⁺ reabsorbed/Ca⁺⁺ reabsorbed changes in favour of the calcium.Lack of Na⁺ on the luminal or the contraluminal side of the tubular wall does not significantly influence the amount of Ca⁺⁺ being reabsorbed. If, however, Na⁺ is missing on both sides the rate of reabsorption of Ca⁺⁺ falls below 10% of the filtered amount.The rate of Ca⁺⁺-reabsorption increases if Ca⁺⁺ is offered on the luminal side only. Presumably this increase is due to a concentration gradient for Ca⁺⁺ which does not exist under physiological conditions. If Ca⁺⁺ is offered on the contraluminal side only, no Ca⁺⁺ appears in the urine. Apparently the tubular epithelium is impermeable for Ca⁺⁺ from the blood to the tubular lumen.The decrease of the reabsorption of Ca⁺⁺ brought about by substances inhibiting the reabsorption of Na⁺ such as Convallatoxin and Furosemid is relatively more marked than the concomitant inhibition of the reabsorption of Na⁺.Since in the experiments with the above mentioned inhibitors the extracellular Na⁺-concentration remains constant, it is concluded that the Ca⁺⁺-reabsorption is coupled with the transport of Na⁺.In principle the Na⁺-dependence of the Ca⁺⁺-reabsorption follows the same pattern as the Na⁺-dependence of the transports of glucose, PAH and urea, thus supporting our contention that the Na⁺-dependence of active transports is a physiological mechanism of general importance.