Novel coagglutination method for serotyping group B streptococci.

A group G streptococcal strain was coated with antibody against six different serotypes (Ia, Ib, II, III, IV, and V) of group B streptococci. The coagglutination patterns of 114 strains of group B streptococci were compared with the serotypes determined after immunoprecipitation. The specificity of the method was 100% and the sensitivity 97%. It was used for the typing of 89 invasive and 101 colonizing isolates. The new method is swift, specific, and highly sensitive. It consumes only minute amounts of antibody.     

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.     

Hand and finger skin temperatures in convective and contact cold exposure

The present study aimed at investigating the spatial variability of skin temperature (T _sk) measured at various points on the hand during convective and cold contact exposure. A group of 8 subjects participated in a study of convective cooling of the hand (60 min) and 20 subjects to contact cooling of the finger pad (5 min). Experiments were carried out in a small climatic chamber into which the hand was inserted. For convective cold exposure,T _sk was measured at seven points on the palmar surface of the fingers of the left hand, one on the palmar surface and one on the dorsal surface of the hand. The air temperature inside the mini-chamber was 0, 4, 10 and 16°C. With the contact cold exposure, the subjects touched at constant pressures an aluminium cube cooled to temperatures of –7, 0 and 7°C in the same mini-chamber. ContactT _sk was measured on the finger pad of the index finger of the left hand. TheT _sk of the proximal phalanx of the index finger (on both palm and back sides), and of the middle phalanx of the little finger was also measured. The variation ofT _sk between the proximal and the distal phalanx of the index finger was between 1.5 to 10°C during the convective cold exposure to an air temperature of 0°C. Considerable gradients persisted between the hand and fingers (from 2 to 17°C at 0°C air temperature) and between the phalanges of the finger (from 0.5 to 11.4°C at 0°C air temperature). The onset of cold induced vasodilatation (CIVD) on different fingers varied from about 5 to 15 min and it did not always appear in every finger. For contact cold exposure, whenT _sk on the contact skin cooled down to nearly 0°C, the temperature at the area close to the contact skin could still be 30°C. Some cases of CIVD were observed in the contact skin area, but not on other measuring points of the same finger. These results indicated that local thermal stimuli were the main determinents of CIVD. Representative hand skin temperature may require five or more measuring points. Our results strongly emphasised a need to consider the large spatial and individual variations in the prediction and modelling of extremity cooling.     

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.     

Quantitative studies on phytohaemagglutinin-induced cytotoxicity by human lymphocytes against homologous cells in tissue culture

The cytotoxic effect of normal human blood lymphocytes on Chang cells in tissue culture was investigated. Cell damage was measured by release of ⁵¹Cr from pre-labelled tissue culture `target' cells. This method was sensitive and rendered highly reproducible results. Released ⁵¹Cr was not re-utilized.

About 25 per cent of the ⁵¹Cr was spontaneously released from labelled Chang cells when incubated for 24 hours at 37°. Lymphocytes at a lymphocyte/Chang cell ratio of 25:1 led to a slight increase of this release. When phytohaemagglutinin was also present, about 50–60 per cent of the isotope appeared in the medium. Under these conditions target cells were significantly damaged within 1 hour. At a lymphocyte/Chang cell ratio of only 1:1, weak cytotoxic effects were also noted after 24 hours of incubation. The results of dose—response experiments suggested that a considerable proportion of the lymphocytes participated in the reaction. Individual variation of the cytotoxic effect of lymphocytes from different donors suggested that it could be related to the degree of histoincompatibility between lymphocytes and Chang cells. Under the present conditions contaminating erythrocytes or granulocytes did not interfere with the cytotoxic action of the lymphocytes.

     

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.     

Computer Simulations of the “Hairy Rod” Model

Summary: Using extensive Molecular Dynamics simulations we study the behavior of very rigid polyelectrolytes with hydrophobic side chains that are known to form cylindrical micelles in aqueous solution. We investigate the stability of such micelles with respect to hydrophobicity, Coulomb interaction, and micellar size. We show that for the parameter range relevant for poly(p‐phenylene sulfonate)s (PPP) one finds a stable finite micellar size close to the experimental parameter region. We also point out that our model has some similarities to DNA solutions with added condensing agents, hinting to the possibility that the size of DNA aggregates is under certain circumstances thermodynamically limited.

DNA‐like morphologies of the polyelectrolytes.     

Cytomegalovirus (CMV) antigenemia assay is more sensitive than shell vial cultures for rapid detection of CMV in polymorphonuclear blood leukocytes.

We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.