Insoluble immune complex-stimulated neutrophil leukotriene B4 production is dependent on Fc gamma RII and Fc gamma RIII and independent of pertussis toxin-sensitive signal transduction pathways.

Leukotriene B4 (LTB4) release initiated by interaction of immune complexes (ICs) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied using well-defined complexes. Immune complexes consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complex) and at five times antigen excess (soluble complex). Incubation of human neutrophils with soluble and insoluble ICs led to the synthesis of LTB4 from endogenous arachidonic acid (AA). LTB4 release induced by ICs was markedly inhibited by monoclonal antibodies against either Fc gamma RII or Fc gamma RIII receptor. Treatment of neutrophils with pertussis toxin significantly inhibited the release of LTB4 induced by soluble ICs. However pertussis toxin treatment minimally inhibited the LTB4 release induced by insoluble ICs. Crosslinking of either Fc gamma RII and Fc gamma RIII receptors on neutrophil surfaces induced LTB4 release. This is the first experimental observation showing that both Fc gamma RII and Fc gamma RIII directly induce neutrophil LTB4 metabolism in the absence of exogenous AA. These studies also suggest the involvement of novel pertussis toxin insensitive signal transduction pathways in insoluble ICs stimulation of neutrophils.     

Interaction of Inflammatory Cells and Oral Microorganisms VII. In Vitro Polymorphonuclear Responses to Viable Bacteria and to Subcellular Components of Avirulent and Virulent Strains of Actinomyces viscosus

Both virulent (V) and avirulent (AV) strains of Actinomyces viscosus T14 are capable of colonizing the oral cavity of gnotobiotic rats, but only T14-V causes destructive periodontal disease. The basis for this difference in in vivo pathogenicity has not been adequately defined. In the present study we compared the capacities of T14-AV and T14-V to provoke in vitro extracellular release of lysosomal constituents from human polymorphonuclear leukocytes (PMNs). In serum-free cultures, viable T14-V but not T14-AV stimulated discharge of PMN lysosomes. The release response was correlated with PMN phagocytic activity; thus, PMNs readily ingested T14-V but not T14-AV. To explain these differences in PMN-bacteria interactions, subcellular fractions of T14-AV or T14-V were incubated with PMNs. A crude, insoluble sonic extract derived from T14-V caused PMN lysosome release, but a similar fraction from T14-AV was inactive. However, following extensive washing and treatment with deoxyribonuclease or sodium dodecyl sulfate, cell wall fractions of T14-AV stimulated lysosome release. These procedures apparently removed an extracellular polysaccharide slime which is synthesized by T14-AV but not by T14-V. There was a significant reduction in the capacities of viable T14-V or cell wall fractions of T14-V or T14-AV to provoke PMN lysosome release when these agents were preincubated with a slime material isolated from T14-AV. This inhibitory influence of slime was overcome by the addition of fresh or heated (56°C, 30 min) serum to the PMN-bacteria cultures. The data suggest a relationship between the abilities of the avirulent and virulent strains of A. viscosus T14 to act as periodontal pathogens in vivo and to serve as stimuli for PMN lysosome release in vitro.     

Use of PCR to study epidemiology of Serratia marcescens isolates in nosocomial infection.

A method to characterize strains of Serratia marcescens based on the PCR amplification of enterobacterial repetitive intergenic consensus sequences has been developed. The PCR fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during an outbreak of pneumonia from seven mechanically ventilated patients, and its result indicated that the outbreak was due to the spread of two epidemic strains. This technique was validated by comparison with rRNA gene restriction analysis. There was complete concordance between these two techniques in discriminating the outbreak-related strains from epidemiologically unrelated isolates. Typing with both biochemical profile and antibiogram profile, though simple, was found to be less reliable than genotyping. The results show that this enterobacterial repetitive intergenic consensus PCR provides a rapid and simple means of typing S. marcescens isolates for epidemiologic studies.     

Interferon in cerebrospinal fluid

Summary Interferon (IFN) was measured in serum and cerebrospinal fluid (CSF) of dogs after experimental (intranasal) infection with different strains of virulent canine distemper virus (CDV). Viral strains employed produce neurological changes in dogs that range from acute inflammatory to subacute, delayed demyelinating encephalomyelitis. With few exceptions, first appearance of serum-IFN correlated with the first elevated body temperature 4 days post-infection (p.i.). By 16 days p.i. IFN had disappeared from the serum of all infected dogs. In contrast, IFN was constantly detectable in CSF in dogs with CDV infection of the central nervous system (CNS). It was first detected 5 days p.i., was continuously detectable during the variable preclinical phase and into the period when signs of acute or delayed encephalomyelitis were evident. Dogs from which CDV would be retrieved from CNS tissue at necropsy always had CSF-IFN (up to 56 days p.i.). In contrast, dogs that recovered from infection, substantiated at necropsy by minimal, resolving CNS lesions and non-detectable virus, had IFN in CSF demonstrable for only a brief post-inoculation period. CSF-IFN appears to be a valid marker for CDV persistence in the canine CNS and may have broader applications.With 3 Figures     

Ovarian stimulation by clomiphene citrate and hMG in combination with cetrorelix acetate for ICSI cycles

BACKGROUND: The introduction of GnRH antagonists such as cetrorelix acetate has made possible the simplification of ovarian stimulation. However, the most effective protocol for their administration has not yet been clearly defined. METHODS: Forty women with male-factor infertility undergoing 40 ICSI cycles were included in the study. Clomiphene citrate at 100 mg a day was given from cycle day 3 through day 7. hMG at 150 IU was given on cycle days 4, 6 and 8, and was adjusted from day 9 according to the follicular and hormone responses. Cetrorelix acetate at 2.5 mg was administered when the leading follicle reached 14 mm. The remaining 0.5 mg was divided into two 0.25 mg injections for possible later use. Serum FSH, LH, estradiol and progesterone levels were measured daily from the day of cetrorelix acetate injection until hCG was given. RESULTS: Serum LH level was suppressed effectively for 4 days. Four patients (10%) needed one or two additional injections of 0.25 mg cetrorelix acetate. No premature LH surge was detected in any of the women treated. Sixteen women became pregnant (40%), of which 14 pregnancies (35%) were ongoing at the time of writing. CONCLUSIONS: This study demonstrates that this new protocol is feasible for couples with male-factor infertility undergoing ICSI.     

Bilateral simultaneous tubal sextuplets: pregnancy after in-vitro fertilization--embryo transfer following salpingectomy

The presence of a damaged tube has been suggested in recent studies to have a negative effect on in-vitro fertilization (IVF) outcome. Performing bilateral salpingectomy prior to IVF to maximize pregnancy rates may also result in unnecessary surgery. This case is also an example of the occurrence of interstitial pregnancy after salpingectomy. This unusual type of ectopic pregnancy must be kept in mind when evaluating a patient suspected of a possible early abnormal gestation after assisted reproductive technolologies.      

Action potential bursts in central snail neurons elicited by d-amphetamine: roles of ionic currents

The roles of the ionic currents in the firing of potential bursts elicited by d-amphetamine in central snail neurons were studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. Oscillations of membrane potential bursts were elicited by d-amphetamine. The action potential bursts elicited by d-amphetamine decreased following intracellular injection of either EDTA or magnesium, or extracellular application of lanthanum. Voltage-clamped studies revealed that d-amphetamine decreased the fast Na(+), Ca(2+) and transient outward K(+) currents of the RP4 neuron. It also decreased the steady-state K(+) current and elicited a negative slope resistance in the steady-state I-V curve between -50 and -10 mV. The amplitude of negative slope resistance was decreased if either Na(+)-free saline or Co(2+)-substituted Ca(2+)-free saline was perfused. d-Amphetamine did not increase the amplitude of the slowly inactivating Ca(2+) current or the persistent Na(+) currents of RP4 neuron. Tetraethylammonium, a blocker of the delayed outward K(+) current, elicited action potential bursts and negative slope resistance in the RP4 neuron, while 4-aminopyridine, an inhibitor of transient outward K(+) current (I(A)), did not.These results demonstrate that the delayed outward K(+) current and the negative slope resistance in steady-state I-V curve elicited by d-amphetamine may be responsible for the action potential bursts in central snail neurons elicited by d-amphetamine.     

Human anti-JC virus serum reacts with native but not denatured JC virus major capsid protein VP1

The immunoreactivity of human anti-JC virus (JCV) serum against the major capsid protein VP1 of JCV was analyzed by Western blot, dot blot, and hemagglutination inhibition (HAI) assays. JCV-positive human serum reacted with native but not denatured JCV major capsid protein VP1, as demonstrated by dot blot and Western blot. Rabbit antiserum raised against native JCV capsid had immunoreactivities similar to those of human anti-JCV serum. These results indicate that the antigenecity of native and denatured JCV VP1 is different. In addition, both JCV-positive human serum and rabbit antiserum raised against native JCV capsid protein inhibited the hemagglutination activity of JCV capsid particles. In contrast, rabbit antiserum raised against denatured JCV VP1 did not inhibit hemagglutination. These findings reveal that denaturation may alter the antigenic epitopes of JCV VP1. Therefore, keeping the JCV capsid protein native appears to be essential for serological or other immunological analyses of the virus.     

Purification of group 2 Dermatophagoides pteronyssinus allergen and prevalence of its specific IgE in asthmatics

Group 2 allergens are a major cause of sensitization in patients allergic to house dust mites. This study was performed to determine the prevalence of hypersensitivity to group 2 allergens (Der p 2) of Dermatophagoides pteronyssinus (Dp) in asthmatic patients in Taiwan. To facilitate the analysis of Der p 2-specific IgE, we raised a panel of monoclonal antibodies (MoAbs) to Der p 2 antigens. Purified Der p 2 was obtained after MoAb affinity column purification. There were 82 asthmatic patients (41 adults and 41 children) with hypersensitivity to Dp who were analyzed for hypersensitivity to Der p 2. All of them were both skin test- and serology test-reactive to Dp. Using purified Der p 2, 87.8% (72/82) of patients had a skin-test-positive reaction. Six adults (6/41) and 4 children (4/41) had negative skin tests for Der p 2. Ten families (both parents and children were asthmatics) of the 82 patients were selected for Der p 2 skin testing and Der p 2-specific IgE determination using immunoblot analysis. Results showed that 90% (18/20) of patients' skin reactions to Der p 2 and serum contained specific IgE to Der p 2. Because 87.8% (85.4% of adults and 90.2% of children) of the asthmatic patients with Dp hypersensitivity were allergic to Der p 2, its role in the pathogenesis of asthma in Taiwan appears to be important. Purified Der p 2 allergens can be further used for allergen skin testing and immunotherapy.     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.