Mutation of the ectodysplasin-A gene results in bone defects in mice.

Anhidrotic ectodermal dysplasia (EDA) is an X-linked, recessive genetic disease characterized by dysfunctional sweat glands, poorly developed teeth, and premature balding in human beings. This disorder results from mutations in the gene for ectodysplasin-A, a type II transmembrane protein with tumour necrosis factor-alpha domains. An animal model of EDA, the Tabby mouse, also has mutations in the ectodysplasin-A gene and defects similar to those of human beings with EDA. In addition to these defects, Tabby mice acquire deformities in the distal portion of their tails at 10-12 weeks of age. Whole-mount staining of the skeleton with Alizarin Red and Alcian Blue revealed that the tail defect resulted from vertebral fractures just distal to the epiphysis. Histological analysis demonstrated that the structure of both the epiphysis and the subepiphyseal zone of the tail vertebrae was dysplastic while the shaft of the diaphysis was relatively normal. The overall structure of the trabecular bone of these animals was examined through 3-dimensional microcomputed tomography of the tibia. This analysis indicated that Tabby mice had a mild increase in the interconnectivity of the intertwined trabecular bone network but that individual trabeculae were relatively normal. Since it has been determined recently that the ectodysplasin-A gene is expressed in the osteoblasts of developing human embryos, it appears likely that this gene plays a role in normal bone development.

Psychopathy and conduct problems in children: II. Implications for subtyping children with conduct problems.

OBJECTIVE: To test whether the presence of callous and unemotional (CU) traits designates a unique subgroup of children with conduct problems that corresponds more closely to adult conceptualizations of psychopathy. METHOD: A clinic-referred sample of 120 children between the ages of 6 and 13 years were assessed using parent and teacher ratings of CU traits, as well as parent and teacher report on a structured interview assessing oppositional defiant disorder (ODD) and conduct disorder (CD) symptoms. RESULTS: A cluster analysis of the ratings of CU traits and ODD/ CD symptoms revealed four clusters of children, two of which had high rates of ODD and CD symptoms. One of these conduct problem clusters also exhibited high levels of CU traits (n = 11). These children had a greater number and variety of conduct problems, a stronger history of police contacts, and a stronger parental history of antisocial personality disorder, despite being of higher intelligence than other children with significant conduct problems (n = 29). CONCLUSION: The presence of CU traits with significant conduct problems seems to designate a unique subgroup of antisocial children who show a very severe pattern of antisocial behavior and who correspond more closely to adult conceptualizations of psychopathy.

Induction of rotavirus-specific cytotoxic T lymphocytes by vaccinia virus recombinants expressing individual rotavirus genes.

We determined the capacity of vaccinia virus recombinants expressing individual rotavirus genes to induce virus-specific cytotoxic T lymphocytes (CTLs) in mice. Mice were orally inoculated with vaccinia virus recombinants containing genes which encode rotavirus outer capsid proteins vp4 or vp7, single-shelled virus proteins vp1, vp2, or vp6, or rotavirus nonstructural proteins NS53, NS35, NS28, or NS26/NS12. We found that (i) the greatest frequencies of virus-specific CTLs were induced by vaccinia virus recombinants expressing vp7, (ii) transport of vp7 beyond the endoplasmic reticulum was not necessary for induction of CTLs, (iii) recombinants expressing vp7 induced CTLs which reacted with different rotavirus serotypes, and (iv) CTLs were induced among both intestinal and nonintestinal lymphocytes after oral inoculation. These findings may be relevant to vaccine strategies which utilize vectors expressing individual rotavirus genes.

Rotavirus-specific helper T cell responses in newborns, infants, children, and adults.

An obstacle to developing a successful rotavirus vaccine has been the inability to consistently correlate the humoral immune response with protection against disease. Transplacental transfer of maternal rotavirus-specific antibodies may obscure the capacity to discriminate an active from a passively acquired humoral immune response in infants. In an attempt to circumvent this problem, an assay was developed to detect rotavirus-specific helper T cells among circulating mononuclear cells. Rotavirus-specific lymphoproliferative responses and rotavirus-specific neutralizing antibody titers in blood were determined in 11 mother/newborn pairs at the time of delivery and in 54 infants, children, and adults ranging in age from 16 days to 40 years. Only 1 of 11 infants tested between 16 days and 6 months of age had detectable rotavirus-specific helper T cell activity whereas 8 of 11 had circulating rotavirus-specific neutralizing antibodies. Acquisition of rotavirus-specific helper T cell activity over the first few years of life correlated with the age at which infants and young children are known to be infected with rotavirus. These findings support the hypothesis that detection of rotavirus-specific lymphoproliferative activity in infants may more accurately determine previous exposure to rotavirus than detection of rotavirus-specific antibodies.

Pathogenesis of intraabdominal abscess formation: abscess-potentiating agents and inhibition of complement-dependent opsonization of abscess-inducing bacteria.

Bacteroides fragilis and Escherichia coli are synergistic in the production of intraabdominal abscesses. However, these bacteria initiate abscess formation only when inoculated with an agent such as autoclaved colonic contents (ACC) or bran (a fiber analogue). The mechanism of action of the abscess-potentiating agent was studied. Opsonins in normal mouse serum were determined for phagocytic killing by murine neutrophils of B. fragilis and E. coli. Opsonization required fixation of complement by the alternative pathway. ACC (0.2 mg/ml) and bran (1.0 mg/ml) inhibited phagocytic killing of Proteus mirabilis in the presence of normal but not immune serum. Assay of the alternative pathway of complement activation indicated that both bacterial components and abscess-potentiating agents in an abscess-inducing mixture activated complement. These findings suggest that abscess-potentiating agents inhibit opsonization and therefore the subsequent phagocytic killing of bacteria in the nonimmune host.

Outer capsid glycoprotein vp7 is recognized by cross-reactive, rotavirus-specific, cytotoxic T lymphocytes.

Cytotoxic T lymphocytes (CTLs) generated in mice orally inoculated with rotaviruses lyse target cells infected with different rotavirus serotypes (cross-reactive CTLs). Using vaccinia virus recombinants expressing individual rotavirus proteins from two different rotavirus serotypes, we found that cross-reactive CTLs recognize target cells expressing outer capsid protein vp7 better than those expressing outer capsid protein vp4 or inner capsid protein vp6. These findings may be relevant to vaccine strategies which include immunization with reassortant rotaviruses or viral or bacterial vectors expressing individual rotavirus proteins. The region or regions of vp7 which are antigenically conserved among different rotavirus serotypes and recognized by cross-reactive CTLs remain to be determined.

Altered neutrophils in mice immune to experimental Naegleria amoebic meningoencephalitis.

Naegleria fowleri is the cause of primary amoebic meningoencephalitis in man. The mouse is considered to be a suitable experimental model for this disease. The data presented shows that blood neutrophils from N. fowleri immune mice (immunised) that had received a 'recall' amoeba antigen had altered responses compared with those from similarly treated normal mice. The neutrophils from immune animals showed increased basal levels of oxygen-dependent respiratory activity, measured by the chemiluminescence response. These neutrophils also showed increased responses to formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), phorbol myristate acetate (PMA), and opsonized N. fowleri. The results are consistent with previous data suggesting that the neutrophil (and its activation) is important in defence against N. fowleri.

Depression of immunity to Naegleria fowleri in mice by selective depletion of neutrophils with a monoclonal antibody.

In an attempt to define the role of neutrophils in immunity to Naegleria fowleri in vivo, we examined the effects of treating immunized (with amoeba culture supernatant antigen) mice with the monoclonal antibody NIMP-R10, which binds to neutrophil complement receptor type 3bi (CR3) and causes selective neutrophil depletion in mice. Mice in the nonimmunized group challenged with amoebae all died by day 12, while 97% in the immunized group survived. By contrast, the immunized group treated with NIMP-R10 showed only 25% survival. The immunized group treated with "control" mouse ascites, WEM-G11, was highly resistant (90% survival). There was a significant neutrophil response in the nasal mucosa and olfactory lobes of immunized, NIMP-R10-treated mice, despite a marked degree of neutropenia similar to that seen in immunized, untreated mice. Nonimmunized mice showed virtually no neutrophil response. Despite this response in the NIMP-R10-treated mice, amoebic proliferation was not depressed, and there was no evidence of neutrophil degranulation or amoebic killing, despite the close apposition of large numbers of neutrophils to amoebae. The results indicate that neutrophils are necessary for the expression of immunity to N. fowleri.

Role of myeloperoxidase in the killing of Naegleria fowleri by lymphokine-altered human neutrophils.

Previously we have shown that human neutrophils treated with conditioned medium from phytohemagglutinin-stimulated mononuclear leukocytes (sCM) in the presence of antisera have amoebicidal properties for Naegleria fowleri, a pathogenic free-living amoeba. The data now presented show that neutrophils which lack myeloperoxidase (MPO) but have a normal oxygen-dependent respiratory burst could not be altered by sCM to express the amoebicidal activity. Catalase inhibited this amoebicidal activity of sCM-treated neutrophils. Various components and products of the neutrophils were examined for effects on naegleriae. A granule extract was found to have no effect at concentrations up to 100-fold that which killed Salmonella minnesota R595. Hydrogen peroxide appeared to have little effect even at 100 microM. However, in the presence of MPO, H2O2 was amoebicidal at 2.5 microM. The generation of amoebicidal activity required the presence of chloride ions. Azide inhibited the effects of the MPO-H2O2-Cl- system. Arginine, a scavenger of hypochlorite, significantly depressed the ability of sCM-treated neutrophils to kill amoebae and also prevented the amoebicidal properties of the MPO-H2O2-halide system. These results suggest that the MPO-H2O2-halide system is important in the killing of naegleriae by sCM-treated neutrophils and that hypochlorite may be the amoebicidal agent.

Roles of antibody and complement in the bactericidal activity of mouse peritoneal exudate neutrophils.

The contributions of complement and antibody to phagocytosis and, as a separate process, intracellular killing of Proteus mirabilis, were investigated using mouse peritoneal exudate neutrophils. Phagocytosis of P. mirabilis was promoted by both immune mouse (IMS) and normal mouse (NMS) sera. Opsonization by IMS promoted significantly greater phagocytosis than did NMS, as did NMS compared with heated IMS (HIMS). The ability of NMS to opsonize P. mirabilis for both phagocytosis and phagocytic killing was diminished by chelation with EGTA and abolished by chelation with EDTA. This suggested that fixation of complement by both alternative and classical pathways provided optimal opsonization of this organism in NMS. In order to study intracellular killing as a process separate from phagocytosis, peritoneal exudate cell suspensions were exposed to P. mirabilis, previously incubated with 1% NMS, 1% IMS, 10% HNMS (heated normal mouse serum) or 10% HIMS, followed by centrifugation of the phagocyte-bacteria mixtures on Percoll density gradients. Populations of neutrophils containing viable intracellular bacteria, and relatively free of extracellular bacteria (less than 7% of total) were recovered in washed suspensions of cells fractionated at densities greater than 1.069 g/ml. For P. mirabilis that had been opsonized with 1% NMS before phagocytosis, the continued presence of extracellular serum was necessary for intracellular killing. NMS stimulated significantly greater intracellular killing than did HNMS, which stimulated some intracellular killing compared with the absence of serum, in which no killing occurred. IMS was similar to NMS in its ability to stimulate intracellular killing. EGTA partially blocked the stimulation of intracellular killing by NMS, and EDTA abolished it. These findings suggested that (as for optimal opsonization) complement activated via both alternative and classical pathways was responsible for optimal stimulation of intracellular killing.

Opsonins in normal mouse serum for the phagocytic killing of Proteus mirabilis by murine neutrophils.

An assay of phagocytic killing by murine neutrophils in homologous serum was used to determine the nature of the opsonins in normal mouse serum for phagocytic killing of Proteus mirabilis. Leucocytes from the peritoneal cavities of mice given an intraperitoneal inoculation of brain-heart infusion broth 3 h previously, phagocytosed and killed P. mirabilis in a 2-h assay in the presence of 10% serum from normal mice. The serum factors supporting phagocytic killing were heat-labile (50 degrees C or 56 degrees C for 30 min) and could be absorbed at 37 degrees C but not 4 degrees C by three different species of Gram-negative bacteria. The tested species of Gram-positive bacterium did not absorb the activity. At the end of the assays, greater than 90% of leucocyte-associated bacteria were associated with neutrophils. Leucocytes from unstimulated peritoneal cavities (less than I% neutrophils) did not kill bacteria in this assay, in contrast to leucocyte suspensions containing up to 98% neutrophils. These findings indicated that the phagocytic killing of P. mirabilis in this assay was mediated by neutrophils, and that complement fixation by the alternative pathway provided necessary opsonins in normal mouse serum.

Local immunity to klebsiella pneumoniae in the lungs of mice.

The development of local immunity to Klebsiella pneumoniae in the lower respiratory tract is described. Immunity to intranasal infection is produced by systemic immunization resulting in high titers of circulating antibody. Protection also follows intranasal immunization with glutaraldehyde-killed organisms. Low levels of antibodies develop in serum after intranasal immunization. IgA antibody can be detected in the serum of these mice whereas little IgA antibody is detected in the serum of mice immunized systemically. Both IgA and IgG antibodies can be found in the pulmonary secretions of locally immunized mice. IgA and IgG from these secretions may be used to passively transfer protection when used to opsonize the infecting dose of K. pneumoniae. Protection mediated by IgA is not due to any interaction of IgA with cells present in the lower respiratory tract during infection, but exerts its protective effect in the upper respiratory tract, thereby preventing the spread of bacterial infection to the lower respiratory tract.

Blood pressure characteristics of a select group of North American males, followed for 20 years.

This report describes the 20-year blood pressure behaviour of 3869 selected young North American males.Initial mean systolic and diastolic pressures were higher than those recorded five years later; after that pressures increased progressively. The effect of initial selection was evident for the first 10 years of exposure.A significant relationship was demonstrated between all initial systolic and diastolic levels and the 20-year blood pressure behaviour. Systolic pressure was not affected by age until age 50 and diastolic until age 45. After that a significant relationship was demonstrated.In 20 years multiple readings >/= 140 and/or >/= 90 mm. Hg were recorded in 26% of the population. Increases in pressure usually extended over many years. Commonly they were labile, fluctuating above and below 140/90 mm. Hg. In a small, clearly defined group, accelerated increases reached high levels in three to 10 years.