Identification of Mycobacterium tuberculosis clinical isolates with altered phagocytosis by human macrophages due to a truncated lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.     

Interaction of the disintegrin and cysteine-rich domains of ADAM12 with integrin alpha7beta1

We describe a novel interaction between the disintegrin and cysteine-rich (DC) domains of ADAM12 and the integrin alpha7beta1. Integrin alpha7beta1 extracted from human embryonic kidney 293 cells transfected with alpha7 cDNA was retained on an affinity column containing immobilized DC domain of ADAM12. 293 cells stably transfected with alpha7 cDNA adhered to DC-coated wells, and this adhesion was partially inhibited by 6A11 integrin alpha7 function-blocking antibody. The X1 and the X2 extracellular splice variants of integrin alpha7 supported equally well adhesion to the DC protein. Integrin alpha7beta1-mediated cell adhesion to DC had different requirements for Mn2+ than adhesion to laminin. Furthermore, integrin alpha7beta1-mediated cell adhesion to laminin, but not to DC, resulted in efficient cell spreading and phosphorylation of focal adhesion kinase (FAK) at Tyr397. We also show that adhesion of L6 myoblasts to DC is mediated in part by the endogenous integrin alpha7beta1 expressed in these cells. Since integrin alpha7 plays an important role in muscle cell growth, stability, and survival, and since ADAM12 has been implicated in muscle development and regeneration, we postulate that the interaction between ADAM12 and integrin alpha7beta1 may be relevant to muscle development, function, and disease. We also conclude that laminin and the DC domain of ADAM12 represent two functional ligands for integrin alpha7beta1, and adhesion to each of these two ligands via integrin alpha7beta1 triggers different cellular responses.     

Bimodal effects of chronically administered neurokinin B (NKB) on in vivo and in vitro cardiovascular responses in female rats

The in vivo cardiovascular effects of acutely administered neurokinin B (NKB) have been attributed both to direct effects on vascular tone and to indirect effects on central neuroendocrine control of the circulation. We proposed: 1) that a modest long-term increase in plasma NKB levels would decrease mean arterial pressure (MAP) due to attenuated peripheral vascular tone, and 2) that chronic high-dose NKB would increase MAP, due to increased sympathetic outflow which would override the peripheral vasodilation. We examined the in vivo and in vitro cardiovascular effects of chronic peripheral NKB. Low- (1.8 nmol/h) or high- (20 nmol/h) dose NKB was infused into conscious female rats bearing telemetric pressure transducers. MAP, heart rate (HR) and the pressor responses to I.V. phenylephrine (PE, 8 microg) and angiotensin II (Ang II, 150 ng) were measured. Concentration-response curves of small mesenteric arteries were constructed to PE using wire myography. Low-dose NKB reduced basal MAP (88+/-2 mm Hg to 83+/-2 mm Hg), did not affect resting HR, reduced the pressor responses to PE, and attenuated the maximal constriction of mesenteric arteries to PE and KCl. By contrast, high-dose NKB increased basal MAP (86+/-1 mm Hg to 89+/-1 mm Hg), increased HR (350+/-3 beats/min to 371+/-3 beats/min), increased the pressor responses to Ang II and, contrary to our hypothesis, increased the maximum contractile responses of mesenteric arteries to PE and KCl. The cardiovascular effects of NKB are thus dose-dependent: whereas chronic low-dose NKB directly modulates vascular tone to reduce blood pressure, chronic high-dose NKB induces an increase in blood pressure through both central (indirect) and peripheral (direct) pathways.     

Variation in the ribosomal internal transcribed spacers and 5.8S rDNA among five species of Acropora (Cnidaria; Scleractinia): patterns of variation consistent with reticulate evolution

The ITS sequences of Acropora spp. are the shortest so far identified in any metazoan and are among the shortest seen in eukaryotes; ITS1 was 70-80 bases, and ITS2 was 100-112 bases. The ITS sequences were also highly variable, but base composition and secondary structure prediction indicate that divergent sequence variants are unlikely to be pseudogenes. The pattern of variation was unusual in several other respects: (1) two distinct ITS2 types were detected in both A. hyacinthus and A. cytherea, species known to hybridize in vitro with high success rates, and a putative intermediate ITS2 form was also detected in A. cytherea; (2) A. valida was found to contain highly (29%) diverged ITS1 variants; and (3) A. longicyathus contained two distinct 5.8S rDNA types. These data are consistent with a reticulate evolutionary history for the genus Acropora.      

Secretory IgA in Urinary Tract Infections

Secretory IgA, measured by radial immunodiffusion, was compared in the urine of children with chronic and recurrent non-obstructive urinary tract infections with that in normal children. IgA, IgG, and IgM were also measured. Absent and low levels of IgA(s) were found in both groups; however, the mean levels of IgA(s) were significantly higher in the infected group compared with normals—3·3 to 0·78 mg./24 hours, respectively. Secretory IgA was found to be locally produced in the bladder. It is suggested that IgA(s) levels reflect an antibody response to infection.     

Auto-dissemination of commercially available fungal pathogens in a laboratory assay for management of the brown dog tick Rhipicephalus sanguineus

Rhipicephalus sanguineus is a canine tick that infests dogs throughout the world and is frequently found in homes and dog kennels. Management of this tick species is complicated by the presence of resistance to commonly utilized acaricides. Fungal formulations could provide a valuable alternative tool for management and are especially relevant indoors where detrimental environmental effects on fungal spores are of less concern. Two commercially available fungal formulations, one containing Metarhizium anisopliae and the other containing Beauveria bassiana, were compared for time to death and sporulation in nymphal ticks exposed for 60 min in treated filter paper packets. Beauveria bassiana exposure killed ticks faster than M. anisopliae exposure and B. bassiana was more likely to sporulate on tick cadavers than M. anisopliae. To determine whether infected ticks could disseminate fungus to their conspecifics, ticks were marked and treated with fungus before being placed with untreated ticks. Fungus was successfully transmitted from treated to untreated ticks. Mortality of ticks exposed to B. bassiana-exposed conspecifics occurred sooner than for those exposed to M. anisopliae-exposed conspecifics, indicating faster dissemination in the former. Therefore, although both formulations resulted in decreased longevity of ticks compared with the controls, the B. bassiana formulation holds the most promise for direct or indirect application with respect to brown dog tick management.     

Regulation of invertase levels in Avena stem segments by gibberellic Acid, sucrose, glucose, and fructose

Gibberellic acid and sucrose play significant roles in the increases in invertase and growth in Avena stem segments. About 80% of invertase is readily solubilized, whereas the rest is in the cell wall fraction. The levels of both types of invertase change in a similar manner in the response to gibberellic acid and sucrose treatment. The work described here was carried out with only the soluble enzyme. In response to a treatment, the level of invertase activity typically follows a pattern of increase followed by decrease; the increase in activity is approximately correlated with the active growth phase, whereas the decrease in activity is initiated when growth of the segments slows. A continuous supply of gibberellic acid retards the decline of enzyme activity. When gibberellic acid was pulsed to the segments treated with or without sucrose, the level of invertase activity increased at least twice as high in the presence of sucrose as in its absence, but the lag period is longer with sucrose present. Cycloheximide treatments effectively abolish the gibberellic acid-promoted growth, and the level of enzyme activity drops rapidly. Decay of invertase activity in response to cycloheximide treatment occurs regardless of gibberellic acid or sucrose treatment or both, and it is generally faster when the inhibitor is administered at the peak of enzyme induction than when given at its rising phase. Pulses with sucrose, glucose, fructose, or glucose + fructose elevate the level of invertase significantly with a lag of about 5 to 10 hours. The increase in invertase activity elicited by a sucrose pulse is about one-third that caused by a gibberellic acid pulse given at a comparable time during mid-phase of enzyme induction, and the lag before the enzyme activity increases is nearly twice as long for sucrose as for gibberellic acid. Moreover, the gibberellic acid pulse results in about three times more growth than the sucrose pulse. Our studies support the view that gibberellic acid, as well as substrate (sucrose) and end products (glucose and fructose), play a significant role in regulating invertase levels in Avena stem tissue, and that such regulation provides a mechanism for increasing the level of soluble saccharides needed for gibberellic acid-promoted growth.     

Acid-sensing ion and epithelial sodium channels do not contribute to the mechanoreceptor component of the exercise pressor reflex

Amiloride, injected into the popliteal artery, has been reported to attenuate the reflex pressor response to static contraction of the triceps surae muscles. Both mechanical and metabolic stimuli arising in contracting skeletal muscle are believed to evoke this effect, which has been named the exercise pressor reflex. Amiloride blocks both acid-sensing ion channels, as well as epithelial sodium channels. Nevertheless, amiloride is thought to block the metabolic stimulus to the reflex, because this agent has been shown to attenuate the reflex pressor response to injection of lactic acid into the arterial supply of skeletal muscle. The possibility exists, however, that amiloride may also block mechanical stimuli evoking the exercise pressor reflex. The mechanical component of the reflex can be assessed by measuring renal sympathetic nerve activity during the first 2-5 s of contraction. During this period of time, the sudden tension developed by contraction onset briskly discharges mechanoreceptors, whereas it has little effect on the discharge of metaboreceptors. We, therefore, examined the effect of amiloride (0.5 microg/kg) injected into the popliteal artery on the renal sympathetic and pressor responses to static contraction of the triceps surae muscles in decerebrated cats. We found that amiloride significantly attenuated the pressor and renal sympathetic responses to contraction; for the latter variable, the attenuation started 10 s after the onset of contraction. Our findings lead us to conclude that acid-sensing ion channels and epithelial sodium channels play little, if any, role in evoking the mechanical component of the exercise pressor reflex.     

Effect of exogenous hormones on the ovulation of primary and secondary oocytes in LT/Sv strain mice

LT/Sv strain mice ovulate both primary and secondary oocytes. These are fertilizable and give rise to digynic triploid and normal diploid conceptuses, respectively. A previous study [Kaufman and Speirs, 1987] had indicated that just over 20% of embryos recovered on the 10th day of gestation from spontaneously ovulating females had a triploid chromosome constitution. This value was considerably lower than might have been expected by extrapolation from earlier studies in which LT/Sv mice had been given exogenous gonadotrophins. In the present study, therefore, cytogenetic analysis of fertilized eggs was performed at the first cleavage mitosis in (1) spontaneously ovulating females mated to F1 hybrid males, and (2) superovulated females mated to similar males. Additional females from group (1) were autopsied on the 10th day of gestation, and the ploidy of embryos isolated at this stage of gestation was determined. Exposure to exogenous gonadotrophins significantly increased the proportion of eggs that were ovulated as primary oocytes (34.4%), compared to the situation observed following spontaneous ovulation (24.4%). All the triploids encountered in both series were of the digynic type and characteristically (for LT/Sv mice) had an oocyte-derived set with 40 chromosomes present, and a sperm-derived set containing 20 chromosomes. Similar numbers of eggs were recovered from spontaneously ovulating females on the 1st and 10th days of gestation, and the incidence of triploidy observed on the 10th day was 22.1%. The influence of exogenous hormones in increasing the “spontaneous” level of triploidy in LT/Sv and in other strains of mice is briefly reviewed.