Mycobacterium tuberculosis ESAT-6 is a leukocidin causing Ca2+ influx, necrosis and neutrophil extracellular trap formation.

Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca(2+) overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca(2+) activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca(2+), stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.

Development of goat embryos after in vitro fertilization and parthenogenetic activation by different methods.

Effective activation protocols that can be used during nuclear transfer investigations in goats need to be developed. We compared the development of IVF goat embryos with those of nonfertilized parthogenetically developing oocytes activated by treatment with either ionomycin or ethanol, both followed by immediate exposure to 6-diethylaminopurine (6-DMAP). Cumulus oocyte complexes (COCs) recovered from abattoir goat ovaries were either matured in a conventional laboratory incubator or placed in pre-equilibrated maturation medium and shipped overnight in a battery-operated dry incubator to another laboratory. Mature COCs were allocated randomly to one of three treatment groups. Group 1 oocytes (n=169 shipped, n=253 not shipped) were fertilized in vitro at 24 h postmaturation (hpm). The remaining COCs were activated at 28 hpm in either ionomycin (Group 2: n=362 shipped, n=202 not shipped), or ethanol (Group 3: n=263 shipped, n=249 not shipped). Activated oocytes were immediately incubated in 6-DMAP for 4 h. Blastocyst development was evaluated on Day 8 post-insemination/activation. Percent cleavage was comparable in shipped and nonshipped oocytes and in all treatment groups. In both shipped and nonshipped oocytes, parthenotes developing from ionomycin- and ethanol-activated oocytes had significantly greater blastocyst development (P<0.01) compared to IVF embryos (28.5 +/- 3.0, 27.4 +/- 2.8, 10.3 +/- 3.0, respectively for the nonshipped oocytes and 9.9 +/- 2.1, 10.3 +/- 2.4, 3.7 +/- 4.7 respectively for the shipped oocytes). Shipped oocytes had lower blastocyst development compared to nonshipped oocytes in the three treatment groups. The mean blastocyst cell number was not statistically different between shipped and nonshipped oocytes or among treatment groups, suggesting that all were equally viable.

Necrotising fasciitis as a complication of botulinum toxin injection.

PURPOSE: To highlight the need for early diagnosis and treatment of the rare condition of necrotising fasciitis as a complication of botulinum toxin injection, and to illustrate that injections in immunocompromised patients carry a rare but serious risk. RESULTS AND METHODS: A case report is presented of an 80-year-old woman suffering from blepharospasm and chronic myeloid leukaemia, who developed necrotising fasciitis 3 days after a botulinum toxin injection. CONCLUSIONS: Chronic debilitating processes such as diabetes, alcoholism and polymyositis have been suggested as predisposing factors in the development of necrotising fasciitis. We believe this is the first reported case of necrotising fasciitis occurring secondary to a botulinum toxin injection. The fact that this infection extended through the fascial planes and led to the death of muscle was, probably, because an inoculum was introduced directly into the muscle at the time of botulinum toxin treatment. This may have led to its deep spread and difficulty in debriding the area. Chronic myeloid leukaemia does not in itself cause significant immunosuppression, but our patient was on anti-proliferative treatment and had a low leucocyte count, which may have been a predisposing factor in this case.