Mycobacterium tuberculosis chaperonin 10 stimulates bone resorption: a potential contributory factor in Pott's disease.

Pott's disease (spinal tuberculosis), a condition characterized by massive resorption of the spinal vertebrae, is one of the most striking pathologies resulting from local infection with Mycobacterium tuberculosis (Mt; Boachie-Adjei, O., and R.G. Squillante. 1996. Orthop. Clin. North Am. 27:95-103). The pathogenesis of Pott's disease is not established. Here we report for the first time that a protein, identified by a monoclonal antibody to be the Mt heat shock protein (Baird, P.N., L.M. Hall, and A.R.M. Coates. 1989. J. Gen. Microbiol. 135:931-939) chaperonin (cpn) 10, is responsible for the osteolytic activity of this bacterium. Recombinant Mt cpn10 is a potent stimulator of bone resorption in bone explant cultures and induces osteoclast recruitment, while inhibiting the proliferation of an osteoblast bone-forming cell line. Furthermore, we have found that synthetic peptides corresponding to sequences within the flexible loop and sequence 65-70 of Mt cpn10 may comprise a single conformational unit which encompasses its potent bone-resorbing activity. Our findings suggest that Mt cpn10 may be a valuable pharmacological target for the clinical therapy of vertebral tuberculosis and possibly other bone diseases.

Effects of extracts prepared from modified porous poly(ether imide) microparticulate absorbers on cytotoxicity, macrophage differentiation and proinflammatory behavior of human monocytic (THP-1) cells.

Remaining uremic toxins in the blood of chronic renal failure patients represent one central challenge in hemodialysis therapies. Highly porous poly(ether imide) (PEI) microparticles have been recently introduced as candidate absorber materials, which show a high absorption capacity for uremic toxins and allow hydrophilic surface modification suitable for minimization of serum protein absorption. In this work, the effects of extracts prepared from PEI microparticles modified by nucleophilic reaction with low molecular weight polyethylene imine (Pei) or potassium hydroxide (KOH), on human monocytic (THP-1) cells are studied. The obtained results suggested that the extracts of Pei and KOH modified PEI absorbers have no negative effect on THP-1 cell viability and do not initiate the critical differentiation towards macrophages. The extracts did not enhance transcript or protein levels of investigated proinflammatory markers in THP-1 cells, namely, TNFµ, MCP1, IL6 and IL8. Based on these findings such modified PEI microparticles should be qualified for further pre-clinical evaluation i.e. in an in vivo animal experiment.

The potent bone-resorbing mediator of Actinobacillus actinomycetemcomitans is homologous to the molecular chaperone GroEL.

Actinobacillus actinomycetemcomitans is a Gram-negative bacterium implicated in the pathology of localized juvenile periodontitis, a condition involving rapid destruction of alveolar bone. We have established that gentle extraction of this bacterium in saline releases a proteinaceous fraction (which we have termed surface-associated material [SAM] which has potent osteolytic activity in the murine calvarial bone resorption assay. Fractionation of the SAM has now revealed that activity is associated with a 62-kD protein. This bone-resorbing activity can be blocked by a monoclonal antibody (raised to the whole bacterium) that is claimed to recognize a protein homologous to the Escherichia coli molecular chaperone GroEL. Purification of this bone-resorbing protein to homogeneity has been achieved by a combination of anion exchange, gel filtration, and ATP-affinity chromatography and the NH2-terminal sequence shows > 95% homology to E. coli GroEL. This GroEL homologue is found in the SAM of A. actinomycetemcomitans but is not found in the osteolytically active SAM from other Gram-negative or Gram-positive bacteria. The GroEL protein from E. coli, but not from Mycobacterium tuberculosis and Mycobacterium leprae, also showed activity in the bone resorption assay. We believe this to be the first observation that a molecular chaperone has the capacity to stimulate the breakdown of connective tissue.

Human esophageal carcinoma cell lines: prostaglandin production, biological properties, and behavior in nude mice.

Prostaglandin production by two continuous human esophageal carcinoma cell lines HCU 18 and HCU 39 derived from poorly and moderately differentiated source tumors, respectively, was investigated. Behavior of both lines in vitro and upon sc inoculation into athymic randombred BALB/c nude mice was also assessed. Approximately half the xenografts induced by HCU 18 cells were invasive, whereas those initiated by HCU 39 cells were all well encapsulated. Although metastases were not detected in mice given injections of HCU 39 cells, metastatic tumors developed in 2 mice inoculated with HCU 18 cells. In addition, HCU 18 cells produced significantly more prostaglandin E (PGE) and prostaglandin F (PGF) than HCU 39 cells. These findings suggest a relationship between PGE and PGF production by human esophageal carcinoma cells and their invasive and metastatic potential in athymic mice.

Influence of nanoporous poly(ether imide) particle extracts on human aortic endothelial cells (HAECs).

Accumulated uremic toxins like indoxyl sulphate, hippuric acid and p-cresyl sulphates in renal failure patients stimulate proinflammatory effects, and consequently kidney and cardiovascular diseases. Low clearance rate of these uremic toxins from the blood of uremic patients by conventional techniques like hemodialysis is due to their strong covalent albumin binding (greater than 95%) and hydrophobic nature, which led to alternatives like usage of hydrophobic adsorber's in removing these toxins from the plasma of kidney patients. Polymers like polyethylene, polyurethane, polymethylmethacrylate, cellophane and polytetrafluoroethylene were already in use as substitutes for metal devices as dialysis membranes. Among new synthetic polymers, one such ideal adsorber material are highly porous microparticles of poly(ether imide) (PEI) with diameters in the range from 50-180µm and a porosity around 88±2% prepared by a spraying and coagulation process.It is essential to make sure that these synthetic polymers should not evoke any inflammatory or apoptotic response during dialysis. Therefore in our study we evaluated in vitro effect of PEI microparticle extracts in human aortic endothelial cells (HEACs) concerning toxicity, inflammation and apoptosis. No cell toxicity was observed when HAECs were treated with PEI extracts and inflammatory/apoptotic markers were not upregulated in presence of PEI extracts. Our results ensure biocompatibility of PEI particles and further hemocompatibility of particles will be tested.

Effect of extracts of poly(ether imide) microparticles on cytotoxicity, ROS generation and proinflammatory effects on human monocytic (THP-1) cells.

Current haemodialysis techniques are not capable to remove efficiently low molecular weight hydrophobic uremic toxins from the blood of patients suffering from chronic renal failure. With respect to the hydrophobic characteristics and the high level of protein binding of these uremic toxins, hydrophobic adsorber materials might be an alternative to remove these substances from the plasma of the chronic kidney disease (CKD) patients. Here nanoporous microparticles prepared from poly(ether imide) (PEI) with an average diameter of 90 ± 30 µm and a porosity around 88 ± 2% prepared by a spraying/coagulation process are considered as candidate adsorber materials. A prerequisite for the clinical application of such particles is their biocompatibility, which can be examined i.e. indirectly in cell culture experiments with the particles' extracts. In this work we studied the effects of aqueous extracts of PEI microparticles on the viability of THP-1 cells, a human leukemia monocytic cell line, as well as their macrophage differentiation, reactive oxygen species (ROS) generation and inflammation.A high cell viability of around 99 ± 18% and 99 ± 5% was observed when THP-1 cells were cultured in the presence of aqueous extracts of the PEI microparticles in medium A and medium B respectively. The obtained microscopic data suggested that PEI particle extracts have no significant effect on cell death, oxidative stress or differentiation to macrophages. It was further found that the investigated proinflammatory markers in THP-1 cells were not up-regulated. These results are promising with regard to the biocompatibility of PEI microparticles and in a next step the hemocompatibility of the microparticles will be examined.

The Escherichia coli chaperonin 60 (groEL) is a potent stimulator of osteoclast formation.

Chaperonins (cpns) are intracellular oligomeric protein complexes that fold and refold proteins in a catalytic manner and aid in the transmembrane transport of cellular proteins. We reported previously that the lipopolysaccharide-free recombinant cpn60 of Escherichia coli (groEL) is able to stimulate the breakdown of murine calvarial bone in culture and showed that such resorption is potently inhibited by an inhibitor of the enzyme cyclo-oxygenase and to a lesser extent by inhibitors of 5-lipoxygenase. In this study, we have investigated the effects of groEL on the resorptive activity and formation of osteoclasts in culture. In low density, osteoclast-containing cultures from neonatal rats incubated for 24 or 96 h on dentine discs, groEL (1-1000 ng/ml) stimulated resorption pit formation up to 4-fold, but this effect was essentially dependent on cell number. Using 12-day cultures of mouse bone marrow to assess osteoclast recruitment, groEL (1-1000 ng/ml) caused a dramatic dose-dependent stimulation of the formation of tartrate-resistant acid phosphatase-positive multinucleated cells and the resorption of the dentine on which bone marrow cells were cultured. Osteoclast formation elicited by groEL was almost completely abolished by indomethacin, an inhibitor of cyclo-oxygenase, but was unaffected by inhibitors of 5-lipoxygenase, suggesting that prostaglandins but not leukotrienes may mediate the action of groEL on osteoclastogenesis. It is possible that bacterial cpn60s such as groEL may play a role in the osteolysis associated with bone infections. Whether endogenous ("self") chaperonins have a role in other bone loss disorders, such as osteoporosis, is an intriguing possibility.

Surface-associated proteins from Staphylococcus aureus demonstrate potent bone resorbing activity.

Staphylococcus aureus infections are associated with rapid bone destruction in conditions such as osteomyelitis, bacterial arthritis, and infected orthopedic implant failure. How this bacterium induces bone destruction has not been defined. In studies of the role of oral Gram-negative bacteria in periodontal pathology, we have established that cell surface-associated proteins (SAPs) are potent stimulators of bone resorption. The surface-associated components from S. aureus have now been isolated and demonstrated to be extremely potent stimulators of bone resorption in the murine calvarial bone resorption assay. Bone resorption appears to be due to proteins, is not the result of contamination with lipoteichoic acid or muramyl dipeptide, and is potently inhibited by indomethacin and can be completely blocked by high concentrations of interleukin-1 receptor antagonist or TN3-19.12, a neutralizing monoclonal antibody to murine TNF. The SAP fraction can stimulate fibroblasts or monocytes to release osteolytic cytokines, but only at high concentrations. Fractionation of the SAPs by high performance liquid chromatography demonstrated that a number of fractions were osteolytically active. The most active contained a heterodimeric protein of molecular weight 32-36 kD. The presence of this osteolytically active surface-associated fraction may account for the bone resorption associated with local infection with S. aureus.

The source of inhibin secretion during the human menstrual cycle.

The source of inhibin secretion during the human menstrual cycle was investigated in two ways. The concentration of inhibin was compared in samples obtained from the ovarian and peripheral veins of 41 women undergoing hysterectomy. In 13 of the women, the corpus luteum was enucleated at operation and the peripheral concentration of inhibin measured at intervals for 24 h. Inhibin was assayed by a heterologous RIA using an antiserum raised against 31 kilodalton bovine inhibin. The concentrations of estradiol and progesterone in the peripheral and ovarian veins were similar to those previously reported. During the early follicular phase, the geometric mean inhibin concentrations were found to be significantly higher in both the right and left ovarian veins than the peripheral vein (180.4 and 157.7 vs. 78.7 U/L: P less than 0.02) but no difference was found in the late follicular phase between the vein draining the dominant ovary and the contralateral ovarian vein (231.1 vs. 193.4 U/L: NS). The inhibin concentrations in the veins draining the ovary bearing a corpus luteum were, however, significantly higher than those in the contralateral ovarian veins during the mid (409.1 vs. 203.6 U/L: P less than 0.02) and late (287.1 vs. 153.2 U/L: P less than 0.01) luteal phases. After enucleation of the corpus luteum, the inhibin concentration fell from the level seen before lutectomy (134.4 U/L) to 80.0 U/L at 24 h (P less than 0.01). This study demonstrates conclusively that the human corpus luteum secretes inhibin. No increase in inhibin secretion was seen from the dominant follicle in the late follicular phase. This casts doubt on the hypothesis that the selective suppression of FSH during the follicular phase is due to inhibin from the dominant follicle.

Evidence for insulin resistance in nonobese patients with polycystic ovarian disease.

In this study seven normal weight Indian patients with polycystic ovarian disease (PCOD) with no evidence of acanthosis nigricans and 7 age- and weight-matched normal Indian women were studied to determine whether PCOD patients were insulin-resistant. While all 14 women had normal glucose tolerance, the PCOD women had significantly higher mean plasma glucose levels at 30 and 60 min and higher mean incremental glucose areas [incremental areas: PCOD, 9.0 +/- 2.2 (+/- SEM); normal women, 4.0 +/- 0.8 mmol/L; P less than 0.05]. Insulin responses were significantly higher in the PCOD compared to normal women (incremental areas: PCOD, 623.8 +/- 78.3; normal women, 226.2 +/- 30.3 microU/mL; P less than 0.001). Both serum testosterone and androstenedione levels correlated with the insulin areas (r = 0.82; P less than 0.001 and r = 0.86; P less than 0.001, respectively). [125I] Insulin binding to erythrocytes revealed decreased maximum specific binding in the PCOD women (6.9 +/- 0.6%) compared to that in normal women (9.2 +/- 0.7%; P less than 0.02). While Scatchard analysis revealed similar receptor numbers, ID50 values demonstrated decreased receptor affinity in the women with PCOD. In conclusion, in the absence of acanthosis nigricans, nonobese patients with PCOD are insulin resistant, and this insulin resistance correlates with the hyperandrogenism.

Amniotic membrane production of prostaglandin F2 alpha is reduced in dysfunctional human labor: results of in vivo and in vitro studies.

Mobilization of arachidonic acid from glycerophospholipids and prostaglandin (PG) release from fetal membranes were studied in women with dysfunctional labor in the absence of cephalopelvic disproportion or fetal malposition. Using superfusion of intact amnion and chorion, we found a slight decrease in PGE and a more significant decrease in PGF release by the amniotic side of the fetal membrane obtained from women with dysfunctional labor compared to that in women with normal labor (PGE: normal labor, 2992 pg/cm2.h; dysfunctional labor, 1846 pg/cm2.h; P less than 0.05; PGF: normal labor, 662 pg/cm2.h; dysfunctional labor, 204 pg/cm2.h; P less than 0.02). Release of both prostanoids was significantly greater from the amniotic side in tissues obtained after labor compared to that in prelabor tissue. Analysis of arachidonic acid (by gas liquid chromatography) and phospholipid content (by two-dimensional thin layer chromatography) confirmed metabolic disposal of arachidonic acid from the amnion after the onset of labor. However, no difference in either phospholipid or phospholipase A2-releasable arachidonic acid of individual phospholipid classes was found in amnion tissue from women with normal and dysfunctional labor, suggesting similar activities of phospholipase A2 in these two groups. The finding of decreased free and phospholipase A2-releasable arachidonic acid of the total lipid extract of the amnion of women with dysfunctional labor could suggest further metabolic exhaustion of the substrate or failure of liberation of this fatty acid from glycerophospholipids by enzymes other than phospholipase A2, such as phospholipase C or diacyl and monoacylglycerolipases.

Amniotic fluid prostanoids in preeclampsia.

Amniotic fluid from 19 patients with preeclampsia was compared with samples from normotensive control subjects with respect to the levels of prostaglandin 6-keto prostaglandin F1 alpha, thromboxane B2, and the ratio of 6-keto prostaglandin F1 alpha to thromboxane B2. The study found no significant differences in the levels of these prostanoids or the ratio of 6-keto prostaglandin F1 alpha to thromboxane B2 (study patients, 2.7 +/- 2.1; control patients, 2.8 +/- 1.9) between groups.

Changes in amniotic fluid prostaglandins with oxytocin-induced labor.

Amniotic fluid prostaglandin levels were measured serially in 15 patients who underwent successful induction of labor and compared with those of patients presenting in spontaneous labor. At comparable cervical dilation the induced group demonstrated significantly lower prostaglandin levels. Four of these patients delivered without any increment in prostaglandins while in the remaining patients increases in prostaglandins followed the attainment of efficient uterine contractions by several hours. These data support the hypothesis that oxytocin acts directly on myometrial cells and not primarily by prior generation of prostaglandin synthesis in the membranes.

Lipid A-associated proteins from periodontopathogenic bacteria induce interleukin-6 production by human gingival fibroblasts and monocytes.

The aim of this study was to determine whether lipid A-associated proteins (LAP) from two periodontopathogenic species of bacteria were able to stimulate interleukin-6 (IL-6) release from human gingival fibroblasts and myelomonocytic cells. LAP and lipopolysaccharide (LPS) were extracted from Porphyromonas gingivalis and Prevotella intermedia and added to cultures of human gingival fibroblasts and mono-mac-6 monocytic cells. Release of IL-6 into the culture supernatants was determined by ELISA. LAP and LPS from Por. gingivalis, but not from Prev. intermedia, stimulated IL-6 release from both cell types in a dose-dependent manner although LPS was less potent than LAP in inducing IL-6 release from the fibroblasts. IL-6 was detectable in cultures of both cell types following stimulation with LAP from Por. gingivalis at a concentration as low as 10 ng/ml. In response to LAP from Prev. intermedia, IL-6 was produced by mono-mac-6 cells but not by fibroblasts. Our results show that bacterial cell wall components other than LPS can induce IL-6 release from cells of the periodontium in vitro. The production of such potent immunomodulatory agents in vivo may contribute to the connective tissue breakdown characteristic of chronic periodontitis.

Serum ribonuclease of normal persons and patients with renal impairment.

Serum ribonuclease of normal persons and of patients with renal impairment was determined with polycytidylic acid as substrate. There was a pronounced rise in the serum ribonuclease of patients with renal impairment. Average serum ribonuclease values of 25 normal persons and 25 patients with renal impairment, respectively were 110 and 2329 units per ml of serum. Serum ribonuclease, because of its unique specificity, stability and abnormal elevation in the sera of patients with renal failure, might serve as an additional indicator in the assessment of renal function.

Serum 5'-nucleotidase of a breast cancer patient.

Serum derived from the breast cancer patient included in this study was found to be a rich source of 5'-nucleotidase. In addition, it also contains nonspecific alkaline phosphatase. The properties of 5'-nucleotidase were studied by eliminating the interference of serum non-specific alkaline phosphatase by the preliminary incubation of serum in glycine-NaOH buffer containing ethylenediamine tetraacetate and magnesium. The enzyme has a pH optimum at 9.5. It is remarkably stable when held at 50 degrees C at pH 7.5, but it readily lost activity in the acid medium at pH 4.2. It hydrolyzes both the ribo- and deoxyribo-nucleoside 5'-phosphates. It has the highest preference for cytidine 5'-phosphate. Adenosine 2'- or 3'- phosphates are refractory to its action.

Polycytidylate hydrolysing ribonuclease variants of human pancreas.

Human pancreatic ribonuclease (RNase) comprises three variants with isoelectric points at pH 4.8, pH 9.3 and pH 9.7. These variants are not artifacts of the purification procedure, since they can be demonstrated in crude pancreatic extracts obtained with mild procedures (4). Although these RNase variants differ in their isoelectric points, they are similar in all the other properties so far studied. With polycytidylic acid as a substrate, their activities are optimal in 0.05M sodium phosphate buffer at pH 6.5. Of the several buffers tested, sodium phosphate buffer yielded maximal enzyme activity, but the concentration of phosphate is highly critical. Borate concentrations of 0.05M and 0.1 M respectively brought about 25% and 50% reductions in enzyme activity. Presence of phosphate in the reaction mixture obviates to a great extent the borate inhibition. These RNase variants are highly specific for the secondary phosphate esters of cytidine 3'-phosphate. They have no measurable activity on polyadenylic and polyguanylic acids and their activity on polyuridylic acid is less than 2% of that on polycytidylic acid. These RNase variants could serve as distinctive biochemical markers for the pancreas.

Male transmission of the gene for isolated gonadotropin-releasing hormone deficiency.

Three black women, daughters of the same father but three unrelated mothers, presented with isolated gonadotropin deficiency (IGD). Clinically, the patients had no midline defects and intact smell and taste senses. Biochemically, the essential feature was very low unstimulated and stimulated follicle-stimulating hormone and luteinizing hormone levels, even after priming with gonadotropin-releasing hormone over a 5-day period. Growth hormone response to insulin-induced hypoglycemia was somewhat blunted, but prolactin, cortisol, and thyroid-stimulating hormone responses were quite normal. All three patients had the 46,XX karyotype; clinical or biochemical aberrations could not be demonstrated in any of the remaining family members. The disorder was, apparently, transmitted by the deceased father, who manifestly did not have an IGD deficiency nor any of the midline stigmata associated with IGD. The mode of inheritance seems most likely to be autosomal dominant with variable penetrance.

A new phosphatase of human platelets.

Human platelets contain a phosphatase, which has a pH optimum at 7.0. Ethylenediamine tetraacetate and citrate at 0.01 mol/l concentration and Mg2+ at 0.002 mol/l concentration respectively bring about 7.7-, 3.6- and 2.1-fold increase in its activity. It is thermolabile. About 70% of its activity is lost when held at 50 degrees C for 10 min at pH 7.5. On electrophoresis through polyacrylamide gel at pH 8.3, about 87.8% of the total activity remained at the origin and the rest migrated toward the anode. Sodium chloride, potassium chloride, sodium phosphate, ouabain in the absence or presence of K+, potassium tartarte, phenylalanine, sodium fluoride have no effect on its activity. Urea at 1 mol/l and 0.5 mol/l concentrations respectively inhibited about 76% and 28% of its activity. It has the highest preference for p-nitrophenyl phosphate. Its Km and Vmax respectively are 5.7 x 10(-4) mol/l and 2.9 mumol . min-1. Its activity on beta-glycerophosphate is about 0.7% of that on p-nitrophenylphosphate. Its activity on ribo- and deoxyribonucleotides ranges from 0.4% to 3.0% of that on p-nitrophenylphosphate. Its maximum stimulation by EDTA, its non-dependence on K+ and its insensitivity to inorganic phosphate and ouabain, make human platelet neutral phosphatase herein described, distinct from the membrane bound neutral phosphtases so far reported.