HLA/Bf haplotypes in a Newfoundland family

HLA/Bf haplotypes were examined in a large three-generation Newfoundland family with a high incidence of Graves' disease. In that family Graves' disease was inherited in association with the haplotype HLA Aw24, Bw39 in some instances and with HLA B8-containing haplotypes in other instances. As all seven members of the family who suffered from Graves' disease were homozygous for the Bf S allele, the study for Bf was uninformative. However, the examination of other HLA/Bf haplotypes disclosed some interesting associations. One-hundred-and-five out of 168 HLA-A, -B, -Bf haplotypes were Bf S. Although numerically deviant, no unusual HLA B/Bf associations were observed. Bf F entered the family only once. A new finding is the association between HLA B27 and Bf S1; the haplotype entered the family once and was passed on to eight family members over three generations. Bf S1 was previously reported in association with HLA B12 or W21. None of these family members had ankylosing spondylitis. The Bf allele F1 entered the family three times, always in association with HLA B18.     

Lectin characterization of gonococci from an outbreak caused by penicillin-resistant Neisseria gonorrhoeae.

A total of 40 Neisseria gonorrhoeae isolates, representing 19 penicillin-resistant isolates (from 8 heterosexual patients and 11 homosexual patients) and 21 penicillin-susceptible isolates (from 15 heterosexual patients and 6 homosexual patients) and obtained from the same geographic area, were examined. Lectin agglutination patterns were based on the reactivity of the isolates with the following 14 lectins: concanavalin A, Lens culinaris, Trichosanthes kinlowii, Griffonia simplicifolia I, Arachis hypogeae (peanut agglutinin), Glycine max (soybean agglutinin), Dolichos bifloris, Griffonia simplicifolia II, Solanum tuberosum (potato starch agglutinin), Triticum vulgaris (wheat germ agglutinin), Limax flavus, Phaseolus vulgaris, Ulex europaeus I, and Lotus tetragonolobus. All isolates were serotyped with monoclonal antibodies specific for gonococcal outer membrane protein I and auxotyped, and the plasmid content was determined. Resistant patient isolates were selected for their decreased penicillin susceptibility, and control isolates were selected for their penicillin susceptibility. Even though the patient isolates demonstrated resistance to penicillin, no phenotypic differences in lectin-grouping patterns were demonstrated between the two study groups; i.e., two predominant lectin groups were observed. No resistance-associated plasmids were detected. All patient isolates were serogroup IB (serovars IB-1, IB-2, and IB-4), whereas 12 of 21 control isolates were serogroup IA (P less than 0.05). Isolates obtained from different anatomical sites in the same patient (cervical and rectal) agreed with regard to lectin patterns and serovars but not auxotypes.     

Human leukocyte antigen-DQ8 transgenic mice: a model to examine the toxicity of aerosolized staphylococcal enterotoxin B

Staphylococcal enterotoxins (SEs) belong to a large group of bacterial exotoxins that cause severe immunopathologies, especially when delivered as an aerosol. SEs elicit the release of lethal amounts of cytokines by binding to major histocompatibility complex (MHC) class II and cross-linking susceptible T-cell receptors. Efforts to develop effective therapeutic strategies to protect against SEs delivered as an aerosol have been hampered by the lack of small animal models that consistently emulate human responses to these toxins. Here, we report that human leukocyte antigen-DQ8 (HLA-DQ8) transgenic (Tg) mice, but not littermate controls, succumbed to lethal shock induced by SEB aerosols without potentiation. Substantial amounts of perivascular edema and inflammatory infiltrates were noted in the lungs of Tg mice, similar to the pathology observed in nonhuman primates exposed by aerosol to SEB. Furthermore, the observed pathologies and lethal shock correlated with an upsurge in proinflammatory cytokine mRNA gene expression in the lungs and spleens, as well as with marked increases in the levels of proinflammatory circulating cytokines in the Tg mice. Unlike the case for littermate controls, telemetric evaluation showed significant hypothermia in Tg mice exposed to lethal doses of SEB. Taken together, these results show that this murine model will allow for the examination of therapeutics and vaccines developed specifically against SEB aerosol exposure and possibly other bacterial superantigens in the context of human MHC class II receptors.     

Restoration of visual orienting into a cortically blind hemifield by reversible deactivation of posterior parietal cortex or the superior colliculus

A contralateral hemineglect of the visual field can be induced by unilateral cooling deactivation of posterior middle suprasylvian (pMS) sulcal cortex of the posterior parietal region, and this neglect can be reversed by additional cooling deactivation of pMS cortex in the opposite hemisphere. The purpose of the present study was to test whether an enduring hemianopia induced by removal of all contiguous visual cortical areas of one hemisphere could be reversed by local cooling of pMS cortex in the opposite hemisphere. Two cats sustained large unilateral ablations of the contiguous visual areas, and cooling loops were placed in the pMS sulcus, and in contact with adjacent area 7 or posterior ectosylvian (PE) cortex of the opposite hemisphere. In both instances cooling of pMS cortex, but neither area 7 nor PE, restored a virtually normal level of orienting performance to stimuli presented anywhere in the previously hemianopic field. The reversal was highly sensitive to the extent of cooling deactivation. In a third cat, cooling deactivation of the superficial layers of the contralateral superior colliculus also restored orienting performance to a cortical ablation-induced hemianopia. This reversal was graded from center-to-periphery in a temperature-dependent manner. Neither the cortical ablation nor any of the cooling deactivations had any impact on an auditory detection and orienting task. The deactivations were localized and confirmed by reduced uptake of radiolabeled 2-deoxyglucose to be limited to the immediate vicinity of each cooling loop. The results are discussed in terms of excitation and disinhibition of visual circuits.     

Endotoxin Binding and Elimination by Monocytes: Secretion of Soluble CD14 Represents an Inducible Mechanism Counteracting Reduced Expression of Membrane CD14 in Patients with Sepsis and in a Patient with Paroxysmal Nocturnal Hemoglobinuria

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% ± 0.8% in healthy subjects to 50.5% ± 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-α) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-α release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 ± 0.4 and 20.1 ± 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-α.     

Generation of the eosinophil chemotactic factor (ECF) from various cell types by melittin

The peptide melittin, the main constituent of bee venom is a potent stimulus for the generation of an eosinophil chemotactic factor (ECF) from human polymorphonuclear neutrophils, rat mast cells and rat peritoneal cells depleted in mast cells. Optimal EFC induction required a sublytic activation of the cells. With each cell type the kinetics of ECF generation were similar in that after an early rise in activity a steep fall off occurred at later times of incubation suggesting a mechanism of inactivation. The induction of ECF by melittin is increased in the presence of calcium. The polar portion of the melittin molecule (aminoacids 20–26) is responsible for the generation of the chemotactic activity. Other peptides of honey bee venom such as the mast cell degranulating peptide (MCD) or apamine do not initiate ECF release. It appears that melittin leads to ECF induction via the phospholipase A₂-arachidonic acid dependent pathway of cell activation. Our data suggests that the lipid mediator ECF can be obtained from phagocytes and mast cells thus indicating the interdependence of inflammatory reactions.     

Frequency of Cytotoxic T Lymphocyte Precursors to Herpes Simplex Virus Type 1 as Determined by Limiting Dilution Analysis

The conditions for establishing a limiting dilution assay to measure cytotoxic T lymphocyte precursors (CTL-P) against herpes simplex virus type 1 (HSV-1) were determined. Analysis by Poisson statistics demonstrated that the estimated frequency of HSV-1-reactive cells in the spleens of normal mice was less than 1/250,000. In contrast, mice immunized previously with infectious HSV-1 demonstrated a CTL-P frequency between 1/3,500 and 1/15,670. The generation of a maximum cytotoxic T lymphocyte response required that mice be primed in vivo with infectious virus. Immunization with inactivated virus either failed to elicit detectable CTL-P frequencies or gave frequencies markedly less than those induced with infectious virus. To obtain positive cultures, the responder cell population had to be exposed to stimulator splenocytes expressing viral antigens. Normal splenocytes without virus or normal splenocytes with T cell growth factor did not result in significant cytotoxicity. Split culture analysis comparing cytotoxicity against syngeneic and allogeneic virus-infected targets provided evidence for specificity, H-2 restriction, and the T cell nature of the CTL-P. It was determined that precursors were eliminated by treatment with anti-Thy 1, Lyt 2.1, or Lyt 1.1, indicating the CTL-P were Lyt 1(+)2(+) cells. Cytotoxicity was reduced after treatment of the responders with anti-Lyt 2 plus complement, which gave further evidence of the T cell nature of the cytotoxic T lymphocytes. These experiments demonstrated the feasibility of using the limiting dilution approach as a highly sensitive and quantitative means to measure the cell-mediated immune response to HSV-1 antigens.     

A differential effect of C5a and C5a des Arg in the induction of pulmonary inflammation.

Earlier studies have shown that C5 fragments induce an inflammatory reaction when instilled into the rabbit lung. Because C5a is rapidly converted to C5a des Arg in vivo, experiments were performed to determine which fragment was most effective in producing pulmonary inflammation in this animal model. C5a des Arg consistently produced marked inflammation. This was characterized by neutrophil accumulation, edema, hemorrhage, fibrin formation, and damage to alveolar epithelium. The time course of the inflammatory reaction initiated by C5a des Arg showed pulmonary vascular sequestration of neutrophils with no intra-alveolar migration at 30 minutes after injection. By 2 hours, interstitial and alveolar neutrophils were numerous, with the accumulation of neutrophils in the alveoli increasing to a maximum at 6 hours. At 24 and 48 hours, the predominant cells were mononuclear (macrophages). By 120 hours, the lesions were resolving. In contrast, at all doses examined, a similar instillation of C5a induced either no inflammation or a milder, more focal response than C5a des Arg. This inability of C5a to initiate inflammation was not apparently due to the generation of inhibitors, since mixtures of C5a and C5a des Arg were phlogistic. A prolonged, intrapulmonary infusion of C5a (20 minutes), in contrast to a bolus instillation (1 minute), did initiate an inflammatory response, which may reflect the conversion of the C5a to C5a des Arg in the lung. This study points out the inflammatory potential of products of complement activation, particularly of the C5 fragment C5a des Arg, when applied to the airway side of the lungs. This inflammatory response raises the possibility that cleavage of intrapulmonary C5 may play an important role in the initiation of pulmonary inflammation.     

Expression of Babesia equi merozoite antigen 1 in insect cells by recombinant baculovirus and evaluation of its diagnostic potential in an enzyme-linked immunosorbent assay

The gene encoding the entire Babesia equi merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiated B. equi-infected horse sera from Babesia caballi-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to B. equi.