Female sex hormones modulate Porphyromonas gingivalis lipopolysaccharide-induced Toll-like receptor signaling in primary human monocytes.

BACKGROUND AND OBJECTIVE: Female sex hormones are elevated and are potential host response modifiers during pregnancy. Modulation of immune responses by estrogen and progesterone may be responsible for periodontal inflammation. Therefore, we aimed to investigate the role of ß-estradiol and progesterone in human monocyte immune responses, at cellular and molecular levels, to identify their role as a possible immunological link between pregnancy and periodontal disease. MATERIAL AND METHODS: Primary human monocytes were purified from human peripheral blood mononuclear cells by adherent method. Expression of Toll-like receptor (TLR) 2, 4 and CD14 was analyzed by flow cytometry. TLR2, TLR4, cyclooxygenase-2 (COX2), nuclear factor-kappa B (NF-κB) and NF-κB inhibitor-alpha mRNA expressions were measured using real-time reverse transcriptase-polymerase chain reaction and prostaglandin E2 secretion was assayed by enzyme-linked immunosorbent assay. NF-κB expression was also examined by immunofluorescence. Western blotting was performed to determine the activation of mitogen-activated protein kinase pathway. RESULTS: We report herein that both ß-estradiol and progesterone significantly reduced TLR2 expression at both protein and mRNA levels but had less of an effect on TLR4 expression in primary human monocytes. We also found that the hormones decreased monocyte cell surface protein expression of CD14. Significantly, ß-estradiol and progesterone dose-dependently downregulated monocyte expression of COX2 mRNA. Pretreatment monocytes with ß-estradiol or progesterone reduced effects of Porphyromonas gingivalis lipopolysaccharide (LPS) on COX2 mRNA expression and decreased prostaglandin E2 secretion by the monocytes. Furthermore, we demonstrated that both ß-estradiol and progesterone inhibited P. gingivalis LPS-induced NF-κB signaling pathway through the upregulation of NF-κB inhibitor-alpha expression. However, neither ß-estradiol nor progesterone altered the phosphorylation of the p38, the extracellular signal-regulated kinase 1/2 and the c-Jun N-terminal activated kinase in P. gingivalis LPS-stimulated monocytes. Thus, the inhibitory effects of these hormones on the response of human monocytes to P. gingivalis LPS appear to be independent on mitogen-activated protein kinase signaling pathway. CONCLUSION: The results of the present study suggest that ß-estradiol and progesterone could influence the immune response of human monocytes to periodontal pathogens and this process may have a role in the clinical manifestations of periodontal disease associated with pregnancy.

Rapid identification of Burkholderia pseudomallei in blood culture supernatants by a coagglutination assay.

In total, 309 blood culture supernatants were tested for the presence of Burkholderia pseudomallei antigen using an in-house coagglutination test prepared by sensitising Cowan I staphylococcal cells with B. pseudomallei polyclonal antiserum. The coagglutination test gave a sensitivity, specificity, positive predictive value and negative predictive value of 100% in comparison with blood culture. A subset of 102 supernatants was also tested for B. pseudomallei antigen using a monoclonal antibody-based latex agglutination test. The sensitivity, specificity, positive predictive value and negative predictive values of this test were 100%, 90%, 75% and 100%, respectively.

Nucleotide sequence of the small subunit ribosomal RNA-encoding gene from Opisthorchis viverrini.

The complete nucleotide (nt) sequence of the small subunit ribosomal RNA-encoding gene of Opisthorchis viverrini reported in this study is the first nt sequence reported for a trematode. The gene is 1992 nt long and has a G + C content of 50.94%. It is made up of alternated constant and variable regions that are similar to the gene organization of other eukaryotes. It is also of interest to note an unexpectedly high degree of sequence homology between O. viverrini and human genes.

Complement activity in children with protein-calorie malnutrition.

Using the hemolytic complement (CH50) assay, we evaluated the complement system of 28 children with severe protein calorie malnutrition (PCM) during their hospital admission and recovery. The mean CH50 activity in children with kwashiorkor was significantly less on hospital days 1 and 4 than in 17 healthy control subjects. On day 8 it rose to normal, and by day 50 it was significantly higher than the controls. The mean CH50 titer of 16 well-nourished febrile children was, in contrast to that of untreated PCM, significantly greater than the healthy controls. Of the children with PCM, 11 (40%) evidenced anticomplementary (AC) activity in their serum on either day or 1 or 4, but only two (7%) had detectable serum AC activity during later convalescence. Significantly, the CH50 titer in a PCM serum correlated inversely to the amount of AC activity in that serum (P less than 0.01). These results indicate that, in children with PCM, the complement system is compromised functionally, and that its repair coincides with intake of adequate diet. The presence of AC activity provides a possible mechanism for depressed complement activity in some untreated PCM children.

Effect of vitamin A and zinc supplementation on the nutriture of children in Northeast Thailand.

Previous surveys suggested that young children in Northeast Thailand may benefit from vitamin A and/or zinc supplementation. One hundred thirty-three children aged 6-13 y with marginal plasma retinol (less than 1.05 mumol/L) and Zn (less than 12.2 mumol/L) concentrations participated in a double-blind study. They were randomly assigned and supplemented with either zinc (25 mg/d), vitamin A (1500 RE/d), zinc plus vitamin A, or placebo for 6 mo. Biochemical indices of vitamin A (plasma vitamin A, retinol-binding protein) and zinc status (plasma zinc, alkaline phosphatase) increased significantly. The children had adequate liver stores of vitamin A (relative dose response less than 20%). Zinc supplementation resulted in an improvement in vision restoration time (VRT) in dim light (dark adaptometry). Vitamin A and zinc synergistically normalized conjunctival epithelium as measured by conjunctival impression cytology (CIC). Both functional indices, VRT and CIC, showed significant correlations with plasma zinc and vitamin A, respectively. The data suggest that functional improvements in populations with suboptimal vitamin A and zinc nutriture can be accomplished by supplementation with less than two times the recommended dietary allowance of these nutrients.

Lymphocyte responsiveness of children supplemented with vitamin A and zinc.

We sought to determine the effect of supplementation with zinc, vitamin A, or a combination of the two on proliferation of T lymphocytes to concanavalin A (ConA), tetanus toxoid (TT), or tuberculin (PPD) of children living in a region endemic for suboptimal vitamin A and zinc intake. The children (n = 140, aged 6-13 y) were randomly assigned and supplemented with either zinc (25 mg/d), vitamin A (1500 mg RE/d), zinc + vitamin A, or placebo for 6 mo. After a baseline blood collection, subjects were boosted with diphtheria-tetanus antigen. Proliferative responsiveness of T lymphocytes to ConA and TT in each treatment group (n = 35) was not different at baseline or postsupplementation. Children supplemented with zinc + vitamin A tended to show higher proliferative responsiveness of T lymphocytes to PPD than did those treated with placebo (P = 0.08). This tendency was observed in females but not in males. Increased zinc and vitamin A intake could result in health benefits for children living in regions endemic for suboptimal micronutrient nutriture.

Secretory and serum IgA in children with protein-calorie malnutrition.

The local immune system of children suffering protein-calorie malnutrition (PCM) was investigated by analyzing the amount of immunoglobulins in the nasal washing on admission, repeatedly during 84 days of hospital therapy, and on follow-up, one to two years later. Although measured concentrations of total protein, IgG, and albumin in nasal washings were reduced in children with PCM, only secretory IgA concentrations were significantly lower (P less than .01) in PCM compared to normal children. Mean secretory IgA concentrations were significantly reduced on admission through hospital day 70 and returned to near normal thereafter. At one to two years after hospital discharge, mean concentrations of secretory IgA in nasal secretions were within normal limits. The concentrations of secretory IgA in nasal washings were lowest at a time when serum IgA was markedly elevated; serum IgA concentrations fell to normal values during dietary treatment. The possible role of secretory IgA deficiency in PCM and infection is discussed.

Antitumor activity of triptolide against cholangiocarcinoma growth in vitro and in hamsters.

One of the diverse biological activities of triptolide, a diterpene from Tripterygium wilfordii, is its antitumor effect. We recently reported its in vitro cytotoxicity against several cultured tumor cell lines. Limited availability of purified fraction has prevented detailed investigation on its antitumor activity. In the present study, we showed by in vitro cytotoxicity assay and in vivo inhibition of tumor growth in hamsters that the triptolide was also highly effective against cholangiocarcinoma, a highly fatal tumor predominantly occurring in developing countries. Its ED50 for these hamster cholangiocarcinoma cell lines was found to be as low as 0.05 microg/ml. The compound was highly potent in the induction of apoptotic death in these tumor cells. DNA fragmentation and disintegrating apoptotic cells could be observed within 24 h of exposure to 0.5 microg/ml triptolide. The compound was tested against the growth of cholangiocarcinoma in a hamster model. A significant growth inhibition (P < 0.05) was noted in triptolide-treated hamsters (each of the 10 animals received 10 injections for a total of 1.2 mg/animal). At the time of sacrifice 1 month after the initial injection, the mean tumor mass of the treated group was only 20-25% of that of the control group.

Production and characterization of monoclonal antibodies against the excretory-secretory antigen of the liver fluke (Opisthorchis viverrini).

Monoclonal antibodies (MoAb) were produced against a major soluble metabolic product (excretory-secretory, ES) of Opisthorchis viverrini. The latter was obtained in a form of spent culture medium in which the adult flukes had been maintained in vitro. The MoAb produced were exclusively associated with either IgG or IgM isotypes. When screened against a panel of parasite antigens by indirect ELISA, these MoAb exhibited three patterns of reactivity. Approximately 50% of the MoAb were highly specific for O. viverrini and another 25% cross-reacted only with Clonorchis sinensis. The remaining 25% cross-reacted extensively with other parasites. Results from radioimmunoprecipitation and immunoblotting experiments showed all MoAb to react with the 89-kDa glycoprotein. By indirect immunofluorescence, these MoAb reacted almost exclusively with the tegumental surface, tegumental cells, cecum and developing miracidium. Different lines of evidence suggest that these MoAb reacted with different epitopes on the same 89-kDa polypeptide carrier.

Monoclonal antibodies to Pseudomonas pseudomallei and their potential for diagnosis of melioidosis.

Monoclonal antibodies (MAbs) specific for Pseudomonas pseudomallei antigens were produced by immunizing BALB/c mice with a crude whole cell extract. Hybrids secreting MAbs specific for P. pseudomallei antigens were identified by an indirect enzyme-linked immunosorbent assay (ELISA) against a panel of crude whole cell extracts from P. pseudomallei, P. cepacia, P. aeruginosa, P. putida, P. alcaligenes, Xanthomonas maltophilia, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Salmonella typhi, S. krefeld, S. enteritidis, Proteus mirabilis, and Staphylococcus aureus. Of the six specific clones, clone 5F8, which was IgM-producing and which reacted with all 56 P. pseudomallei isolates, was selected for further characterization and evaluation of its possible diagnostic potential. Results obtained from the indirect ELISA against various P. pseudomallei antigens, from direct bacterial agglutination, and from immunofluorescence tests suggested that 5F8 reacted with the surface envelope, and probably specifically with an epitope of the lipopolysaccharide. The antibody could be readily used to identify P. pseudomallei in primary culture or in a simulated hemoculture. The antibody was also used to prepare an affinity-purified antigen for use in an indirect ELISA that was highly sensitive and specific for the detection of circulating antibody in patients with acute septicemic melioidosis.

Characterization of humoral immune response in the serum and bile of patients with opisthorchiasis and its application in immunodiagnosis.

The humoral immune response in patients with opisthorchiasis was investigated using an enzyme-linked immunosorbent assay. IgG antibody reactive with Opisthorchis viverrini antigens was present in the serum of all patients. The infection also stimulated specific IgA and IgE antibody responses in most patients and, in practically all patients, there was a marked increase of total IgE. There was a moderate but significant correlation between serum IgG antibody level and severity of infection as judged from the quantity of eggs in the stool of the patients. There was also a significant elevation of antibody in the bile and serum of O. viverrini-infected patients who also had biliary obstruction. Analysis of paired samples from individual patients showed that while IgG was the predominant class of antibody in the serum of all patients, IgA was present at approximately the same level as IgG or higher in the bile of many patients. In addition to IgA and IgG antibodies, IgE antibody was also detectable in 50% of the bile samples. The high level of IgA antibody in the bile together with its presence in association with the secretory component suggested a selective transport and/or local production of IgA antibody by the hepatobiliary system of these patients.

Comparison of the polymerase chain reaction and serologic tests for diagnosis of septicemic melioidosis.

For diagnosis of melioidosis, we compared polymerase chain reaction (PCR)-based DNA detection and three serologic methods with the culture method currently used as gold standard. The diagnostic values of the serologic methods were evaluated in 130 patients. All these patients resided in an endemic area. An enzyme-linked immunosorbent assay (ELISA) gave slightly higher specificity (86.2%) than a dot immunoassay (DOT) (85.3%), but was superior to an indirect hemagglutination assay (IHA) (79.8%). The sensitivities of the DOT (85.7%) and ELISA (71.4%) were considerably higher than that of IHA (61.9%). However, the PCR was the most sensitive (95.2%) and specific (91.7%). Nevertheless, DOT and ELISA are more practical for local hospitals. With the high negative predictive value of both the ELISA (94.0%) and DOT (96.9%) in a high prevalence area, clearly these methods can rule out most of the non-melioidosis patients.

Diagnostic value of an antibody enzyme-linked immunosorbent assay using affinity-purified antigen in an area endemic for melioidosis.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in southeast Asia. The septicemic form of melioidosis is the leading cause of death from nonhospital-acquired septicemia in the northeastern part of Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. The present study was undertaken to evaluate the use of enzyme-linked immunosorbent assays based on an immunoaffinity-purified antigen for detecting specific IgG and IgM antibodies to this organism as a rapid serodiagnostic method for melioidosis. The diagnostic value of these tests was evaluated in an actual clinical situation in an area endemic for melioidosis. The specificity of specific IgG test (82.5%) and the specific IgM test (81.8%) were significantly better than that of the indirect hemagglutination (IHA) test (74.7%). The sensitivity of the specific IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the specific IgM test (63.5%). Specific IgG antibody was detected in a majority of septicemic melioidosis (87.8%), as well as in localized forms (82.6%). The specific IgG test was also better than the specific IgM test and the IHA test in identifying acute melioidosis cases in the first five days after admission. In addition, the IgG antibody level to this antigen remained high over a period of more than five years in those who had recovered from melioidosis and remained clinically free of the disease. These results indicate that the detection of specific IgG antibody is clinically useful for the diagnosis of acute melioidosis in an endemic area.

Detection of Opisthorchis viverrini by monoclonal antibody-based ELISA and DNA hybridization.

Monoclonal antibody-based enzyme-linked immunosorbent assay and DNA hybridization techniques were developed and evaluated for their potential in the detection of Opisthorchis viverrini infection in humans. A mixture of three IgG1 monoclonal antibodies (MAb) specific for the 89 kDa metabolic product of O. viverrini was captured on a microtiter plate by rabbit anti-mouse IgG and used in a sandwich ELISA for the detection of parasite antigen. The 89 kD component bound to the MAb was detected with biotinylated rabbit IgG antibody to O. viverrini metabolic products. As little as 0.05-0.1 ng of the antigen could be detected by this technique. A specific O. viverrini DNA probe constructed from a repetitive DNA segment containing 340 base pairs was used in a dot blot hybridization for the detection of parasite DNA. The labeled probe constructed as such could detect DNA released from as few as five O. viverrini eggs. Both methods were specific for O. viverrini and their sensitivity was comparable with that of the classical parasitological technic.

Evaluation of a monoclonal antibody-based enzyme linked immunosorbent assay for the diagnosis of Opisthorchis viverrini infection in an endemic area.

A monoclonal antibody-based enzyme-linked immunosorbent assay (MAb-ELISA) was evaluated for its potential in the diagnosis of opisthorchiasis in an area endemic for Opisthorchis viverrini infection. The method, based on the detection of the 89-kD O. viverrini metabolic antigen in the feces (coproantigen), was previously estimated to be sensitive enough to detect antigen excreted by a single mature fluke. In the present study, fecal specimens from 207 apparently healthy villagers in northeastern Thailand were analyzed in a double-blind test for the presence of O. viverrini eggs by microscopic examination and for antigen by MAb-ELISA. The microscopic examination was carefully done to minimize false-positive results due to eggs of Lecithodendriid trematodes. The specimens were divided into six groups based on the number of eggs per gram of feces, namely, egg negative, 1-500, 501-1,500, 1,501-3,000, 3,001-6,000, and more than 6,000. The results showed that the ELISA is sufficiently sensitive and specific for the diagnosis of O. viverrini infection. The slightly higher rate of coproantigen positive by the ELISA compared with microscopic examination may reflect lower specificity of the ELISA or its higher sensitivity over microscopic examination in detecting light infections. Different lines of evidence presented here support the latter explanation.

Short report: evaluation of a monoclonal antibody-based latex agglutination test for rapid diagnosis of septicemic melioidosis.

A monoclonal antibody (MAb)-based latex agglutination (MAb-LA) test was developed to rapidly identify Burkholderia pseudomallei in hemoculture of patients with septicemic melioidosis. The method was evaluated in a clinical situation on 396 hemocultures positive for bacterial growth, of which 75 cultures were positive for B. pseudomallei by conventional biochemical tests. The sensitivity and specificity of the MAb-LA test were 95% and 100%, respectively. The positive and negative predictive values were 100% and 99%. The method is highly reliable and suitable for rapid diagnosis of septicemic melioidosis, reducing the time normally required from a minimum of 3-4 days by conventional methods to less than 30 hr. Most of these 30 hr are involved in growing up enough bacteria to perform the MAb-LA test, which itself takes only 1 min.

Monoclonal antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia.

A monoclonal antibody-based latex agglutination (MAb-LA) test was employed for the rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. These patients were admitted to 12 hospitals in the northeastern part of Thailand which is a region known to be endemic for melioidosis. Blood samples were collected and immediately added to the blood culture bottles which were incubated in either automated (five hospitals) or manual (seven hospitals) culture systems. Of a total of 1369 culture-positive specimens, 204 specimens were culture-positive for B. pseudomallei. Of those, 194 (95%) were positive by MAb-LA and the type of blood culture system did not affect positivity rates. The performance of the MAb-LA test on these specimens was highly satisfactory compared with culture detection and confirmation by biochemical test, with 95.1% sensitivity, 99.7% specificity and 98.8% and 99.2% for positive and negative predictive values, respectively. The method described is highly reproducible, simple to perform even by inexperienced laboratory personnel and does not require expensive or elaborate equipment.

Quantitative analysis of immunoglobulins and albumin in secretion of female reproductive tract.

Total protein, IgG, secretory IgA (SIgA), IgA, IgM, and albumin were quantitatively analyzed in 115 cervical fluid specimens from healthy, adult women. Although albumin was the most predominant protein among those that were analyzed (17.2% of total protein), IgG was the major immunoglobulin in this secretion (8.7%). A highly significant correlation between the levels of IgG and albumin and a mean IgG-albumin ratio similar to that of serum suggest that both proteins originate from the circulation. Although the main type of IgA was of the secretory type (4.4%), serum type IgA (smaller than 1.0%) was close to 2. The mean levels of IgG and albumin (but not of SIgA) of the postpartum group were significantly higher than those of the normal subjects. The number of children, the current method of contraception, and present and past local infections had no effect on the immunoglobulins and albumin in this secretion when the mean levels of these components were analyzed and compared with the "normal" values.

IgE responses in human gnathostomiasis.

Specific IgE antibody levels in the serum of patients with proven gnathostomiasis and in those with intermittent cutaneous migratory swellings were determined by the enzyme-linked immunosorbent assay (ELISA) using aqueous extracts of Gnathostoma spinigerum infective larvae as antigen. There was not only an elevation of specific IgE antibody levels but also a marked increase of total IgE in the serum of these patients. The mean levels of specific IgE antibody in both groups of patients were significantly higher than that of healthy controls (P less than 0.01). Only one of the 50 serum specimens tested had an ELISA reading that fell within the mean + 2 standard deviations of the control group, suggesting that the method would be useful with a high degree of reliability in confirming a clinical diagnosis of gnathostomiasis in humans. Compared with healthy controls, there was almost a 10-fold rise (P less than 0.01) in the total IgE in the serum of these patients, indicating that G. spinigerum infection potentiates IgE production similarly to many other nematode parasites.

Immunology and molecular biology of Opisthorchis viverrini infection.

Opisthorchiasis is the major public health problems in Laos PDR and Thailand. The disease becomes chronic and persists for many years, leading to hepatobiliary disease and cholangiocarcinoma. Less severe manifestations include cholangitis, chronic cholecystitis and cholelithiasis. A significant degree of humoral and cell mediated immune responses to the parasite can be detected both in patients and animal models. The patients IgG levels appear to correlate with gall bladder size and dysfunction and correlated significantly with opisthorchis egg count and decrease after treatment. However, the possible significance of these immune responses to protective immunity is presently unknown. The development of immunodiagnostic method for Opisthorchis viverrini detection has been attempted. The components with molecular weight >116, 89, 78 and 20 kDa appear to be specifically associated with the somatic extract of adult fluke. The 89 kDa protein is the most prominent component found in the in vitro culture fluid of adult worms and the metacercarial extract that can be a candidate with significant immunodiagnostic potential. Highly specific and sensitive monoclonal antibodies for O. viverrini antigens were prepared to detect parasite antigens in stool and antibody in serum. Information regarding the molecular approaches of O. viverrini is very limited. The genome of O. viverrini has neither CpG nor A methylated as found in other parasites. The total length O. viverrini ribosomal DNA is approximately 13 kb. and the presence of a highly repeated DNA specific for the parasite was demonstrated. A O. viverrini specific DNA probe was constructed and PCR based detection with high specificity for amplification of the repeated sequences is performed to detect the presence of eggs' DNA in stool samples in comparison with classical methods.