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.     

Immune Responses to Bile-Tolerant Helicobacter Species in Patients with Chronic Liver Diseases, a Randomized Population Group, and Healthy Blood Donors

Bile-tolerant Helicobacter species such as Helicobacter pullorum, Helicobacter bilis, and Helicobacter hepaticus are associated with hepatic disorders in animals and may be involved in the pathogenesis of chronic liver diseases (CLD) in humans. Antibody responses to cell surface proteins of H. pullorum, H. bilis, and H. hepaticus in serum samples from patients with CLD, a randomized population group, and healthy blood donors were evaluated by using enzyme linked immunosorbent assay (ELISA). The results were compared with the antibody responses to Helicobacter pylori. For analysis of a possible cross-reactivity between bile-tolerant Helicobacter species and H. pylori, sera from a subpopulation of each group were absorbed with a whole-cell extract of H. pylori and retested by ELISA. Results before absorption showed that the mean value of the ELISA units for H. pullorum was significantly higher in patients with CLD than in healthy blood donors (P = 0.01). Antibody reactivity to cell surface protein of H. hepaticus was also significantly higher in the CLD patients than in the healthy blood donors and the population group (P = 0.005 and P = 0.002, respectively). Following the absorption, antibody responses to H. pullorum decreased significantly in all three groups (P = 0.0001 for CLD patients, P = 0.0005 for the population group, and P < 0.0001 for the blood donors), indicating that cross-reactivity between H. pylori and other Helicobacter spp. occurs. The antibody responses to H. hepaticus and H. bilis in CLD patients remained high following absorption experiments compared to ELISA results before absorption. The significance of this finding requires further investigations.     

A quantitative ELISA for antigen-specific IgG subclasses using equivalence dilutions of anti-kappa and anti-subclass specific secondary reagents Application to the study of the murine immune response against the capsular polysaccharide of Neisseria meningitidis serogroup B

We have developed an enzyme-linked immunosorbent assay (ELISA) to measure murine antigen-specific IgG antibodies of defined subclass using precalibrated equivalence dilutions of anti-κ (in the standard) and each anti-IgG subclass-specific polyclonal secondary antibody (in the test sample). The calibration of secondary reagents could be carried out easily with a set of monoclonal antibodies (MoAbs) specific for all IgG subclasses. These MoAbs do not require purification or standardization. In addition the MoAbs can be of different antigenic specificity. Once the equivalence dilutions have been determined, they can be applied in a quantitative ELISA using the same antigen in the standard and sample, and using only one IgG subclass standard for the determination of all the IgG subclasses. The method is easy to standardize for many antigenic systems. It is particularly useful when the only standard available is one standardized MoAb of the appropriate specificity, and it could be adapted to use with standard polyclonal antibodies having a known content of total antigen-specific IgG bearing κ chains but unknown IgG subclass composition. The use of this method to quantitate IgG specific for the capsular polysaccharide of Neisseria meningitidis serogroup B (CpsB) gave highly reproducible measures with an interbatch CV of 5–6% similar for all IgG subclasses and low detection limits ranging from 0.3 ng/well for IgG3 to 0.8 ng/well for IgG2a. The IgG subclass response observed after immunization with live meningococci was mainly IgG2a (74%) and IgG2b (18%). Hyperimmunization modified this IgG distribution to one of mainly IgG3 (62%) and IgG1 (28%) which was maintained in the response to a single immunization 4 weeks later, possibly indicating the generation of resting B cells during continuous stimulation.     

Informed consent and knee arthroscopies: an evaluation of patient understanding and satisfaction

Fifty patients who had been admitted to a public hospital in Australia for an elective knee arthroscopy were asked to complete a detailed questionnaire prior to theatre, designed to evaluate patient understanding and satisfaction of the informed consent process. While patients generally felt that they received an appropriate amount of information on the nature of their injury and the actual operative procedure, little information was given on possible complications and post-operative care. This study clearly indicates that patients are dissatisfied when they perceive a lack of basic information being given. The findings are of some concern as they suggest that the majority of consents gained in this study were not truly 'informed'. However, recognition by medical staff of the areas that appear to be poorly explained to patients enables these issues to be addressed. This in turn is likely to improve patient understanding of and satisfaction with the consent process.     

Bovine NK cells can produce gamma interferon in response to the secreted mycobacterial proteins ESAT-6 and MPP14 but not in response to MPB70

Bovine NK cells have recently been characterized and the present study describes the interaction between NK cells, antigen-presenting cells, and secreted mycobacterial proteins. Gamma interferon (IFN-gamma) production by NK cells was seen in approximately 30% of noninfected calves in response to the Mycobacterium tuberculosis complex-specific protein ESAT-6, MPP14 from Mycobacterium avium subsp. paratuberculosis, and purified protein derivative (PPD) from M. tuberculosis. In contrast, no response was induced by MPB70, which is another M. tuberculosis complex-specific secreted antigen. The production of IFN-gamma by NK cells in whole blood in response to ESAT-6 and MPP14 was demonstrated using intracellular staining together with surface labeling for the NK cell-specific receptor, NKp46, or CD3. Furthermore, the depletion of NK cells from peripheral blood mononuclear cells completely abolished the IFN-gamma production. The response was mediated through stimulation of adherent cells and was largely independent of contact between adherent cells and the NK cells. Neutralization of interleukin-12 only partly inhibited IFN-gamma production, showing that other cytokines were also involved. The demonstration of NK cell-mediated IFN-gamma production in young cattle provides an explanation for the nonspecific IFN-gamma response frequently encountered in young cattle when using the IFN-gamma test in diagnosis of mycobacterial infections.     

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.