Biomedical subjects
J Mertsola
Publications and source records attributed to J Mertsola.
Endotoxin concentrations in cerebrospinal fluid correlate with clinical severity and neurologic outcome of Haemophilus influenzae type B meningitis.
Endotoxin concentrations were measured in paired samples of cerebrospinal fluid from 38 patients with Haemophilus influenzae type b meningitis. On admission, the median concentration of endotoxin in cerebrospinal fluid was 104 ng/mL and decreased rapidly in follow-up samples. From 17 to 48 hours after admission, 50% of the patients had concentrations of less than 1 ng/mL. Endotoxin concentrations correlated significantly with concentrations of interleukin 1 beta, protein, and glucose in cerebrospinal fluid, duration of secondary fever, and neurologic abnormalities during hospitalization and on follow-up examinations. Twenty-eight percent of patients with endotoxin concentrations of 100 ng/mL or more on admission had long-term complications, compared with none of those with lower endotoxin concentrations (relative risk, 2.31; 95% confidence interval, 1.53 to 3.48). These results indicate that quantitation of endotoxin in cerebrospinal fluid could be a valuable aid in identifying those children at increased risk of complications during Haemophilus influenzae type b meningitis and provide additional evidence that the Haemophilus influenzae type b meningitis lipo-oligosaccharide is important in the pathogenesis of meningitis.
Cytokines in the pathogenesis of bacterial meningitis.
Adjunctive dexamethasone therapy to antibiotic therapy for bacterial meningitis has been shown in several studies to be beneficial. The roles of interleukin 1 beta and tumour necrosis factor alpha (TNF-alpha) in bacterial meningitis and in an experimental rabbit model are reviewed; both cytokines play a pivotal role in induction of meningitis. Using dexamethasone with the initial dose of antibiotics caused a decrease in TNF-alpha and modulated the inflammatory response. Early use of adjunctive dexamethasone is important in therapy of bacterial meningitis.
Detection of experimental Haemophilus influenzae type b bacteremia and endotoxemia by means of an immunolimulus assay.
The rapid quantitation of bacteria in blood was achieved by using a novel assay method for gram-negative bacterial lipopolysaccharide (endotoxin, LPS). The assay involves the capture of specific LPS onto microtiter plates by means of monoclonal antibodies directed against the oligosaccharide region of the LPS, followed by detection of the bound LPS by a chromogenic Limulus amebocyte lysate (LAL) system. This immunolimulus (IML) assay combines the specificity of monoclonal antibodies with the sensitivity of the LAL system to achieve the first specific, sensitive quantitation of bioactive endotoxin in plasma. In the animal model tested, Haemophilus influenzae type b (Hib) bacteremia in infant rats, there was a strong correlation between IML results and the concentration of Hib colony-forming units in blood samples (r = .845, P less than .001). Using antibodies with appropriate specificities, this approach should be useful for rapid detection of a wide range of gram-negative bacteria and endotoxins in blood.
In vivo and in vitro expression of Haemophilus influenzae type b lipooligosaccharide epitopes.
An indirect immunofluorescence system involving monoclonal antibodies (MAbs) directed against surface epitopes of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) was used to examine individual Hib cells in cerebrospinal fluid (CSF) from infants with Hib meningitis. In four of five CSF samples studied, 100% of the bacteria bound at least one LOS-directed MAb. When the bacteria from these CSF samples were grown in broth, most of these cells lost some or all of their ability to bind the MAb(s) that were bound by the same organisms present in human CSF. When in vitro-grown cells were used for intracisternal injection of rabbits, the populations of Hib cells observed in rabbit CSF after the development of meningitis generally resembled those of the respective broth-grown inocula in terms of their LOS antigenic characteristics. Hib cell populations recovered in infant rat CSF after intranasal challenge again had LOS antigenic characteristics similar to those of the in vitro-grown challenge inocula. These results indicate that a population of Hib cells growing in the infected human host may be quite different, with regard to its LOS antigenic characteristics, from the same Hib strain growing in vitro or in vivo in animal models.
Molecular cloning of a gene involved in lipooligosaccharide biosynthesis and virulence expression by Haemophilus influenzae type B.
A wild-type Haemophilus influenzae type b (Hib) genomic DNA library was constructed in the plasmid shuttle vector pGJB103. A virulence-deficient lipooligosaccharide (LOS) mutant of Hib was used as a recipient for genetic transformation to screen this Hib genomic DNA library for genes involved in LOS expression. A recombinant plasmid containing a 7.8 kb PstI fragment of Hib DNA was shown to transform this LOS mutant to reactivity with a monoclonal antibody (mAb) specific for a wild-type LOS epitope. Transformation of two different virulence-deficient LOS mutants with a 4.4 kb BglII fragment of this recombinant plasmid yielded transformants which expressed LOS that bound the wild-type LOS-specific mAb and yielded profiles in sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis different from those of the original LOS mutants. These transformants with structurally altered LOS molecules also exhibited increased virulence in an animal model for invasive Hib disease. The virulence-transforming ability was further localized to a 1.8 kb BglII-AlwNI fragment of the Hib DNA insert. Nucleotide sequence analysis indicated the presence of a single large open reading frame within this fragment. This open reading frame contained 19 consecutive repeats of the tetramer CAAT near the 5' end. Linker insertion mutagenesis was used to demonstrate directly the involvement of this open reading frame in both LOS biosynthesis and virulence expression by Hib.
Recurrent wheezy bronchitis and viral respiratory infections.
Fifty four patients aged from 1 to 6 years who had had recurrent attacks of wheezy bronchitis were prospectively followed up for three months to find out if there was an association between different viral respiratory infections and episodes of wheezing. Of the 115 episodes of upper or lower respiratory tract symptoms, virus or Mycoplasma pneumoniae infection were diagnosed in 52 (45%). Thirty four of rhinoviruses. The patients had an average of 2.1 episodes of respiratory tract symptoms the total mean (SD) duration of which was 30 (2) days of the 92 days that followed. Wheezing occurred during 76 (66%) of the 115 episodes and during a third of these the patient was admitted to hospital because of severe dyspnoea. Wheezing started a mean (SD) of 43 (7) hours after the first symptoms of respiratory infection and persisted for 3.8 (4.2) days in patients in whom virus infection was diagnosed. The incidence of wheezing was not associated with IgE mediated atopy, with positive virological tests, or with fever during virus infection, but was associated with parental smoking and more than one sibling.
Tumor necrosis factor alpha/cachectin and interleukin 1 beta initiate meningeal inflammation.
Although previous studies using human cytokines in rabbits and rats have provided evidence of the participation of tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) in the meningeal inflammatory cascade, the results obtained by several groups of investigators have been discordant or, at times, contradictory. In the present study, homologous cytokines were applied to the rabbit meningitis model. Intracisternal administration of 10(2)-10(5) IU of purified rabbit TNF-alpha (RaTNF-alpha) produced significant cerebrospinal fluid (CSF) inflammation. A similar response was observed after intracisternal inoculation of 5-200 ng of rabbit recombinant IL-1 beta (rrIL-1 beta). Preincubation of these two mediators with their specific antibodies resulted in an almost complete suppression of the CSF inflammatory response. In animals with Haemophilus influenzae type b lipooligosaccharide-induced meningitis, intracisternal administration of anti-rrIL-1 beta, anti-RaTNF-alpha, or both resulted in a significant modulation of meningeal inflammation. Simultaneous administration of 10(3) IU of RaTNF-alpha and 5 ng of rrIL-1 beta resulted in a synergistic inflammatory response manifested by a more rapid and significantly increased influx of white blood cells into the CSF compared with results after each cytokine given alone. These data provide evidence for a seminal role of TNF-alpha and IL-1 beta in the initial events of meningeal inflammation.
Detection of interleukin 1 beta but not tumor necrosis factor-alpha in cerebrospinal fluid of children with aseptic meningitis.
Tumor necrosis factor-alpha and interleukin 1 beta have been shown to be mediators of meningeal inflammation in animal models of bacterial meningitis. The presence of both cytokines in cerebrospinal fluid (CSF) of patients with bacterial meningitis has been documented recently. In this study, we measured concentrations of interleukin 1 beta and tumor necrosis factor-alpha in CSF samples from 36 patients with nonbacterial (aseptic) meningitis, 13 of whom had culture-proved enteroviral meningitis, and from 14 control patients. None of the samples from patients with aseptic meningitis and from the controls had detectable tumor necrosis factor activity in CSF. Thirty-two (89%) of 36 patients with aseptic meningitis had detectable interleukin 1 beta in CSF (mean +/- SEM, 48 +/- 11 pg/mL). These concentrations were significantly smaller than those previously reported in patients with bacterial meningitis (944 +/- 128 pg/mL). Only 2 of the 14 control patients had detectable CSF interleukin 1 beta concentrations of 21 and 42 pg/mL. A significant correlation was evident between interleukin-1 beta concentrations and white blood cell counts in the CSF of patients with aseptic meningitis. Our data suggest that the initial events of CSF inflammation in children with aseptic meningitis are different than those in patients with bacterial meningitis, and the participation of these two cytokines, especially tumor necrosis factor-alpha, is less critical to the process.
Molecular pathophysiology of bacterial meningitis: current concepts and therapeutic implications.
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Serologic diagnosis of pertussis: evaluation of pertussis toxin and other antigens in enzyme-linked immunosorbent assay.
IgM, IgA, and IgG antibodies to Bordetella pertussis were measured in paired sera from 34 patients who were culture-positive for pertussis by enzyme-linked immunosorbent assay (ELISA) with disrupted B. pertussis bacteria, purified pertussis toxin, or outer membrane proteins (OMP) as antigens. Paired sera from 50 patients with other respiratory infections were used as controls. The sensitivities of the assays from paired sera were 61%, 90%, and 90% and specificities were 98%, 92%, and 72%, respectively. Of the patients culture-positive for pertussis, 68% had positive levels of antibody to pertussis toxin antigen in their first serum samples, obtained at the same time as samples for culture. Infants had antibody responses to pertussis toxin antigen, in contrast to weak antibody responses measured by B. pertussis antigen. The results from this study indicate that ELISA, especially measuring pertussis toxin IgA, is a valuable additional tool for diagnosing pertussis and can be used as a complementary test with cultures.
Pentoxifylline modulates meningeal inflammation in experimental bacterial meningitis.
Pentoxifylline has been shown to decrease endotoxin-induced tumor necrosis factor alpha production and reverse the inflammatory actions of interleukin-1 (IL-1) and tumor necrosis factor on leukocyte function. Because of the potential role of this cytokine-leukocyte interaction in the pathogenesis of bacterial meningitis, we investigated the ability of pentoxifylline to modulate meningeal inflammation in the rabbit meningitis model. Pentoxifylline treatment (initially an intravenous injection of 20 mg/kg followed by 6 mg/kg per h) started 20 min before intracisternal injection of 20 ng of Haemophilus influenzae type b lipooligosaccharide (endotoxin) reduced significantly concentrations in cerebrospinal fluid of leukocytes (P less than 0.0001), protein (P less than 0.001), and lactate (P less than 0.001) during the 9-h infusion compared with values in intravenous-saline-treated rabbits. When pentoxifylline was given 1 h after H. influenzae type b endotoxin, the mean peak lactate and leukocyte concentrations in cerebrospinal fluid were significantly lower than those in control animals. Pentoxifylline also significantly decreased lactate and protein concentrations (P less than 0.05) and tended to diminish leukocyte counts (P = 0.08) compared with results in control animals after antibiotic-induced release of endotoxin in animals with H. influenzae meningitis. In this regard, dexamethasone was superior to pentoxifylline and no synergism was observed when the drugs were combined. Additionally, pentoxifylline attenuated meningeal inflammatory changes induced by intracisternal inoculation of 10 ng of rabbit recombinant IL-1 beta compared with results in either dexamethasone- or saline-treated animals. We conclude that pentoxifylline is effective in this animal model in modulating the meningeal inflammatory response following intracisternal inoculation of H. influenzae type b endotoxin or organisms or rabbit recombinant IL-1beta.
Effect of mutations in lipooligosaccharide biosynthesis genes on virulence of Haemophilus influenzae type b.
Chemical mutagenesis techniques and genetic transformation methods were used to construct isogenic mutants of Haemophilus influenzae type b (Hib) defective in the ability to synthesize lipooligosaccharide (LOS). A mutant (17B) which expressed a LOS molecule with an altered oligosaccharide was less virulent than the wild-type parent strain, as determined by measurement of the ability of these strains to produce bacteremia in infant rats after intranasal challenge. Further mutagenesis of this mutant strain yielded two new mutants with different LOS phenotypes. Mutant 7A was slightly sensitive to the bactericidal activity present in normal infant rat serum and was even less virulent than its immediate parent strain (17B) in the intranasal challenge model. However, both mutants 17B and 7A could produce bacteremia and meningitis when introduced into infant rats by the intraperitoneal route. The other LOS mutant (14A) derived from mutant 17B exhibited a level of virulence equivalent to that of the original wild-type strain. Genetic transformation of wild-type chromosomal DNA into the essentially avirulent mutant 7A and selection of transformants on the basis of their LOS antigenic characteristics resulted in the sequential restoration of full virulence to this mutant. These findings suggest that LOS is involved on at least two different levels in the ability of Hib to produce invasive disease in the infant rat model. Changes in LOS phenotype can independently affect the ability of Hib to produce bacteremia after intranasal challenge and the sensitivity of Hib to killing by normal infant rat serum. These results reinforce the significance of Hib LOS in the expression of virulence by this pathogen.
Specific detection of Haemophilus influenzae type b lipooligosaccharide by immunoassay.
Three monoclonal antibody (MAb)-based immunoassays were developed for specific detection of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS). (i) Hib LOS was captured onto microtiter plates by polyclonal Hib-directed antibodies and detected with MAbs to the oligosaccharide component of Hib LOS in an enzyme-linked immunosorbent assay, (ii) The high affinity of polymyxin B for lipid A was used to bind Hib LOS to microtiter wells, and the oligosaccharide-specific MAbs were used as the detection system in the polymyxin B-MAb assay. (iii) Hib LOS solubilized in detergent was captured by MAbs, and the immobilized LOS was detected with a chromogenic Limulus amebocyte lysate method in the immunolimulus assay. Endotoxin concentrations were measured in in vitro samples and cerebrospinal fluid samples from rabbits with experimental Hib meningitis. The results were compared with those obtained with the standard chromogenic Limulus amebocyte lysate assay. There were significant correlations between the results of all four assays. These new immunoassays provide methods for specific detection of Hib LOS in laboratory fluids and in research involving quantification of Hib endotoxin in experimental animal models.
Clinical role of respiratory virus infection in acute otitis media.
The clinical characteristics of acute otitis media in relation to coexisting respiratory virus infection were studied in a 1-year prospective study of 363 children with acute otitis media. Respiratory viruses were detected using virus isolation and virus antigen detection in nasopharyngeal specimens of 42% of the patients at the time of diagnosis. Rhinovirus (24%) and respiratory syncytial virus (13%) were the two most common viruses detected. Adenovirus, parainfluenza viruses, and coronavirus OC43 were found less frequently. The mean duration of preceding symptoms was 5.9 days before the diagnosis of acute otitis media. Ninety-four percent of the children had symptoms of upper respiratory tract infection. Fever was reported in 55% and earache in 47% of cases. Patients with respiratory syncytial virus infection had fever, cough, and vomiting significantly more often than patients with rhinovirus infection or virus-negative patients. No significant differences were found in the appearance of the tympanic membrane and outcome of illness between virus-negative and virus-positive patients with acute otitis. Most patients respond well to antimicrobial therapy despite the coexisting viral infection. If the symptoms of infection persist, they can be due to the underlying viral infection, and viral diagnostics preferably with rapid methods may be clinically useful in these patients.
Specific detection of Haemophilus influenzae type b lipooligosaccharide by a polymyxin B monoclonal antibody assay.
A monoclonal antibody (MAb)-based enzyme immunoassay was developed for detection of Haemophilus influenzae type b (Hib) lipooligosaccharides (LOS). The high affinity of polymyxin B for lipid A was used to bind the Hib LOS to microtiter wells. The immobilized LOS was detected with MAbs directed against the oligosaccharide component of Hib endotoxin. Hib LOS concentrations were measured in in vitro samples and in cerebrospinal fluid (CSF) sample obtained from rabbits with experimental Hib meningitis. The sensitivity of the assay was 1 ng LOS/ml sample and the results obtained with this assay correlated significantly with those obtained with the standard Limulus amebocyte lysate assay. This new assay provides a method for specific detection of Hib LOS in CSF samples and in aqueous laboratory fluids. This general methodology should also be useful for experimental research involving specific LPS/LOS molecules.
Modulation of inflammation and cachectin activity in relation to treatment of experimental Hemophilus influenzae type b meningitis.
By use of an experimental lapin model, the effect of ceftriaxone therapy on inflammation during Hemophilus influenzae type b (Hib) meningitis was evaluated. Meningitis was induced by intracisternal inoculation of 2 x 10(4) to 2 x 10(5) colony-forming units of Hib. Administration of ceftriaxone 6 h after infection provoked rapid bacterial lysis associated with greatly increased concentrations of bacteria-free endotoxin (lipooligosaccharide) and tumor necrosis factor alpha (TNF) in the cerebrospinal fluid (CSF). CSF TNF activity peaked 2 h after initiation of antibiotic therapy (24.4 +/- 2 ng/ml), was significantly higher than that in untreated controls (1.4 +/- 1.1 ng/ml; P less than .05), and was associated with a substantially increased inflammatory response as reflected by higher CSF white blood cell count (24,500 +/- 8,151 vs. 1,920 +/- 644 in untreated controls; P less than .05), lower glucose, and higher protein and lactate concentrations. Simultaneous administration of dexamethasone with antibiotic therapy resulted in a significant reduction in CSF TNF activity that was associated with a substantial reduction in CSF white blood cell count. These data suggest that initiation of ceftriaxone therapy in experimental Hib meningitis exacerbates the meningeal inflammatory response, which can be modulated by concurrent dexamethasone administration.
Increased endotoxin and interleukin-1 beta concentrations in cerebrospinal fluid of infants with coliform meningitis and ventriculitis associated with intraventricular gentamicin therapy.
Intraventricular gentamicin therapy in infants with gram-negative enteric bacillary meningitis and ventriculitis is associated with increased mortality. Therefore, endotoxin, interleukin-1 beta, and cachectin (tumor necrosis factor) concentrations in ventricular cerebrospinal fluid from 21 infants (11 received intravenous antibiotics alone and 10 received intraventricular gentamicin also) were determined and correlated with outcome and other ventricular cerebrospinal fluid indexes of inflammation. Mean interleukin-1 beta concentrations in ventricular cerebrospinal fluid correlated significantly with adverse outcome and with mean concentrations of endotoxin, white blood cells, and protein and inversely with glucose concentrations. Mean and peak endotoxin and interleukin-1 beta concentrations were significantly higher in infants who received intraventricular gentamicin and intravenous antibiotics than in infants given intravenous antibiotics alone. Intraventricular gentamicin may have caused release of endotoxin from gram-negative bacilli in ventricular cerebrospinal fluid, resulting in increased interleukin-1 beta concentrations and inflammation, which could have contributed to the poor outcome in these patients.