Pregnancy outcomes of antiphospholipid syndrome: In a low resource South Asian setting.

PROBLEM: Antiphospholipid syndrome is associated with recurrent pregnancy loss, and specific treatment improves pregnancy outcome. Laboratory diagnosis is limited in South Asia. We assessed management outcomes of definite/probable antiphospholipid syndrome treated at a tertiary centre in Sri Lanka. METHOD: Descriptive cross-sectional study of pregnancy outcomes with heparin and aspirin therapy. OUTCOME MEASURES: miscarriage, intrauterine death and live birth when compared to previous untreated pregnancies. RESULTS: Of 646 gestations in 145 women, 146 (22.6%) received specific treatment. In the preceding pregnancies without specific treatment, the rates of miscarriage, late fetal loss, stillbirth and live birth were 60%, 26%, 8% and 7%, respectively. Following specific treatment with low-dose aspirin ± low-molecular weight heparin in 146 pregnancies (145 women), the rates of miscarriage, late fetal loss, stillbirth and live birth were 14%, 10%, 3% and 74%, respectively. Mean birth weight was 2.54 ± 0.62 kg, preterm births complicated 32 (29.6%) with a mean gestational age at delivery 33.7 ± 2.6 weeks, with three neonatal deaths. Maternal complications were: pre-eclampsia 16 (10.9%), gestational diabetes 28 (19.2%), antepartum haemorrhage in 1 patient. Only 73/145 (50.3%) women had laboratory confirmation of antiphospholipid syndrome, while others were treated empirically. Live births in diagnosed vs. empiric treatment - 80.8% vs. 67.1%. CONCLUSION: Pregnant women with clinical antiphospholipid syndrome when treated with low-dose aspirin and heparin, the live birth rate of 7% in the previous pregnancy resulted in live births of 74% in a resource limited South Asian setting.

Porphyromonas gingivalis proteinases as virulence determinants in progression of periodontal diseases.

Porphyromonas gingivalis, one of the major causative agents of periodontal diseases, produces large amounts of arginine- and lysine-specific cysteine proteinases in cell-associated and secretory forms, which are now referred to as Arg-gingipain (Rgp) and Lys-gingipain (Kgp), respectively. A number of studies have revealed that these proteinases are closely associated with the periodontopathogenesis of this bacterium: destruction of periodontal connective tissues, disruption of host defense mechanisms, and development and maintenance of inflammation in periodontal pockets. With respect to the physiology of the bacterium, Rgp and Kgp are indispensable for it to obtain nutrients from the environment, since it cannot utilize saccharides as carbon/energy sources for growth and totally depends on peptides and amino acids that are provided from environmental proteins by Rgp and Kgp. Furthermore, proteolytic activities of Rgp and Kgp contribute to processing/maturation of various cell-surface proteins of P. gingivalis, such as fimA fimbrilin (a subunit of major fimbriae), 75-kDa protein (a subunit of minor fimbriae), hemagglutinins, and the hemoglobin receptor protein, which are important for the bacterium to colonize and proliferate in the gingival crevice and to invade the periodontium. These findings strongly indicate critical roles of Rgp and Kgp in the virulence of P. gingivalis.

Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis. Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA.

Porphyromonas gingivalis produces arginine-specific cysteine proteinase (Arg-gingipain, RGP) and lysine-specific cysteine proteinase (Lys-gingipain, KGP) in the extracellular and cell-associated forms. Two separate genes (rgpA and rgpB) and a single gene (kgp) have been found to encode RGP and KGP, respectively. We constructed rgpA rgpB kgp triple mutants by homologous recombination with cloned rgp and kgp DNA interrupted by drug resistance gene markers. The triple mutants showed no RGP or KGP activity in either cell extracts or culture supernatants. The culture supernatants of the triple mutants grown in a rich medium had no proteolytic activity toward bovine serum albumin or gelatin derived from human type I collagen. Moreover, the mutants did not grow in a defined medium containing bovine serum albumin as the sole carbon/energy source. These results indicate that the proteolytic activity of P. gingivalis toward bovine serum albumin and gelatin derived from human type I collagen appears to be attributable to RGP and KGP. The hemagglutinin gene hagA of P. gingivalis possesses the adhesin domain regions responsible for hemagglutination and hemoglobin binding that are also located in the C-terminal regions of rgpA and kgp. A rgpA kgp hagA triple mutant constructed in this study exhibited no hemagglutination using sheep erythrocytes or hemoglobin binding activity, as determined by a solid-phase binding assay with horseradish peroxidase-conjugated human hemoglobin, indicating that the adhesin domains seem to be particularly important for P. gingivalis cells to agglutinate erythrocytes and bind hemoglobin, leading to heme acquisition.

Fertility in dairy cows managed for calving intervals of 12, 15 or 18 months.

Effects on reproduction in a total of 135 dairy cows managed for calving intervals of 12, 15 or 18 months (72, 38 and 25 cows respectively) were studied. The cows were of the Swedish Red and White Breed (SRB) and the Swedish Freisian Breed (SLB) and were housed in 2 different herds with 3 different management systems (tied, loose, and tied but milked in a milking parlour; mixed). The cows in one of the herds (48 cows) were assigned for milking either 2 times or 3 times a day. When comparing conception rate at 1st insemination (AI) and the percentage of cows finally pregnant, we found no significant differences between the 3 calving interval groups, however, a tendency for a higher conception rate with a 15 months' interval compared with a 12 months' interval was found in one of the herds (50% vs 41.5%). The percentage of finally pregnant animals varied between 81% and 100%, but this variation was mainly attributed to the herd rather than calving interval group. A significantly higher percentage of cows was treated for anoestrous in the 12-month group than in the 15-month group in one of the herds (28.6% vs. 5.3%). The frequency of ovulations with external heat signs increased with ovulation number up to the 4th ovulation and thereafter remained stable. No significant difference was found in number of AIs required per conception with respect to calving intervals, breeds, or milking frequency groups. However, cows milked 3 times a day had a significantly longer interval from the 1st AI to conception compared with cows milked 2 times a day (45.8 days vs 17.6 days, p < .01). Cows kept loose exhibited 1st ovulatory oestrous, approximately 2 weeks earlier (55.9 days vs 69.7 days, p < .05) than their herd mates kept tied. In conclusion, our study shows that lengthening the calving interval to 15 or 18 months may have a positive influence on reproduction in terms of less need for treatments of ovarian disorders and higher conception rates. Our results also indicate that milking 3 times a day may have negative effects, and keeping cows in a loose-housing management system may have positive effects on ovarian function.

Involvement of a lysine-specific cysteine proteinase in hemoglobin adsorption and heme accumulation by Porphyromonas gingivalis.

The oral anaerobic bacterium Porphyromonas gingivalis, a major pathogen of advanced adult periodontitis, produces a novel class of cysteine proteinases in both cell-associated and secretory forms. A lysine-specific cysteine proteinase (Lys-gingipain, KGP), as well as an arginine-specific cysteine proteinase (Arg-gingipain), is a major trypsin-like proteinase of the organism. Recent studies indicate that the secreted KGP is implicated in the destruction of periodontal tissue and the disruption of host defense mechanisms. In this study, we have constructed a KGP-deficient mutant to determine whether the cell-associated KGP is important for pathophysiology of the organism. Although the mutant retained the strong ability to disrupt the bactericidal activity of polymorphonuclear leukocytes, its hemagglutination activity was reduced to about one-half that observed with the wild-type strain. More important, the mutant did not form black-pigmented colonies on blood agar plates, indicating the defect of hemoglobin adsorption and heme accumulation. Immunoblot analysis showed that the expression of a 19-kDa hemoglobin receptor protein, which is thought to be responsible for hemoglobin binding by the organism, was greatly retarded in this mutant. The mutant also showed a marked decrease in the ability to degrade fibrinogen. These results suggest the possible involvement of KGP in the hemoglobin binding and heme accumulation of the organism and in the bleeding tendency in periodontal pockets.

Haemoglobin receptor protein is intragenically encoded by the cysteine proteinase-encoding genes and the haemagglutinin-encoding gene of Porphyromonas gingivalis.

The obligately anaerobic bacterium Porphyromonas gingivalis produces characteristic black-pigmented colonies on blood agar. It is thought that the black pigmentation is caused by haem accumulation and is related to virulence of the microorganism. P. gingivalis cells expressed a prominent 19 kDa protein when grown on blood agar plates. Analysis of its N-terminal amino acid sequence indicated that the 19 kDa protein was encoded by an internal region (HGP15 domain) of an arginine-specific cysteine proteinase (Arg-gingipain, RGP)-encoding gene (rgp1) and was also present in genes for lysine-specific cysteine proteinases (prtP and kgp) and a haemagglutinin (hagA) of P. gingivalis. The HGP15 domain protein was purified from an HGP15-overproducing Escherichia coli and was found to have the ability to bind to haemoglobin in a pH-dependent manner. The anti-HGP15 antiserum reacted with the 19 kDa haemoglobin-binding protein in the envelope of P. gingivalis. P. gingivalis wild-type strain showed pH-dependent haemoglobin adsorption, whereas its non-pigmented mutants that produced no HGP15-related proteins showed deficiency in haemoglobin adsorption. These results strongly indicate a close relationship among HGP15 production, haemoglobin adsorption and haem accumulation of P. gingivalis.