Molecular studies on ancient M. tuberculosis and M. leprae: methods of pathogen and host DNA analysis.

Humans have evolved alongside infectious diseases for millennia. Despite the efforts to reduce their incidence, infectious diseases still pose a tremendous threat to the world population. Fast development of molecular techniques and increasing risk of new epidemics have resulted in several studies that look to the past in order to investigate the origin and evolution of infectious diseases. Tuberculosis and leprosy have become frequent targets of such studies, owing to the persistence of their molecular biomarkers in ancient material and the characteristic skeletal lesions each disease may cause. This review examines the molecular methods used to screen for the presence of M. tuberculosis and M. leprae ancient DNA (aDNA) and their differentiation in ancient human remains. Examples of recent studies, mainly from Europe, that employ the newest techniques of molecular analysis are also described. Moreover, we present a specific approach based on assessing the likely immunological profile of historic populations, in order to further elucidate the influence of M. tuberculosis and M. leprae on historical human populations.

Use of lean principals to improve flow of patients with fractured neck of femur--the HOPE study.

We describe the implementation of a care pathway for patients with fractured neck of femur (NOF) using Lean and Six Sigma principles. After introduction of the Lean pathway, 32 patients out a total of 86 (37%) with fractured NOF were admitted to the Trauma Ward within 4 hours of presentation to the hospital; prior to implementation this was 16 patients out of a total of 59 (27%). Post-Lean an earlier mean theatre start time of 8.40am was achieved, resulting in a 38 minute increase in daily theatre time. An additional 52 patients (12%) received surgery within 24 hours of admission, resulting in 1 night length of stay reduction. Lean methodology proved an effective method to guide change resulting in an improved journey for the patient and significant workflow gains.

Insights gained from palaeomicrobiology into ancient and modern tuberculosis.

The direct detection of ancient Mycobacterium tuberculosis molecular biomarkers has profoundly changed our understanding of the disease in ancient and historical times. Initially, diagnosis was based on visual changes to skeletal human remains, supplemented by radiological examination. The introduction of biomolecular methods has enabled the specific identification of tuberculosis in human tissues, and has expanded our knowledge of the palaeopathological changes associated with the disease. We now realize that the incidence of past tuberculosis was greater than previously estimated, as M. tuberculosis biomarkers can be found in calcified and non-calcified tissues with non-specific or no visible pathological changes. Modern concepts of the origin and evolution of M. tuberculosis are informed by the detection of lineages of known location and date.

New skeletal tuberculosis cases in past populations from Western Hungary (Transdanubia).

The distribution, antiquity and epidemiology of tuberculosis (TB) have previously been studied in osteoarchaeological material in the eastern part of Hungary, mainly on the Great Plain. The purpose of this study is to map the occurrence of skeletal TB in different centuries in the western part of Hungary, Transdanubia, and to present new cases we have found. Palaeopathological analysis was carried out using macroscopic observation supported by radiographic and molecular methods. A large human osteoarchaeological sample (n=5684) from Transdanubian archaeological sites ranging from the 2nd to the 18th centuries served as a source of material. Spinal TB was observed in seven individuals (in three specimens with Pott's disease two of which also had cold abscess) and hip TB was assumed in one case. The results of DNA for Mycobacterium tuberculosis were positive in seven of the eight cases identified by paleopathology, and negative in the assumed case of hip TB. However, the molecular results are consistent with highly fragmented DNA, which limited further analysis. Based on the present study and previously published cases, osteotuberculosis was found in Transdanubia mainly during the 9th-13th centuries. However, there are no signs of TB in many other 9th-13th century sites, even in those that lie geographically close to those where osteotuberculous cases were found. This may be due to a true absence of TB caused by the different living conditions, way of life, or origin of these populations. An alternative explanation is that TB was present in some individuals with no typical paleopathology, but that death occurred before skeletal morphological features could develop.

Cyclosporin immunosuppression of sheep: pharmacokinetics and allograft survival.

A chronically immunosuppressed sheep model was established using a regimen of cyclosporin A (CsA; 2-3mg/kg twice daily) and ketoconazole (10mg/kg twice daily). Blood CsA concentrations reached a steady-state after 17 days of treatment. The clearance of CsA decreased from a mean (95% CI) of 9.47 (6.2-12.7)ml/min/kg after a single (first) dose (3mg/kg i.v.) to 1.62 (1.38-1.86)ml/min/kg after 18 days of CsA (3mg/kg i.v. twice daily) co-administration with ketoconazole. These data indicated that the combination of CsA and ketoconazole could be used to give stable high concentrations of CsA in the sheep. Using this regimen in the sheep, the long-term survival of skin allografts was monitored as an indicator of effective immunosuppression. CsA in blood was measured daily and CsA dose adjusted to various target concentration ranges. Provided that the trough concentration of blood CsA was maintained between 1500-2500 mg/l, long-term healthy skin allografts were maintained on the sheep without significant adverse effects on haematological or biochemical parameters.

Mycobacterium tuberculosis complex DNA from an extinct bison dated 17,000 years before the present.

In order to assess the presence of tuberculosis in Pleistocene bison and the origin of tuberculosis in North America, 2 separate DNA extractions were performed by 2 separate laboratories on samples from the metacarpal of an extinct long-horned bison that was radiocarbon dated at 17,870+/-230 years before present and that had pathological changes suggestive of tuberculosis. Polymerase chain reaction amplification isolated fragments of tuberculosis DNA, which were sequenced, and on which spoligotyping was also performed to help determine its relationship to the various members of the Mycobacterium tuberculosis complex. Extensive precautions against contamination with modern M. tuberculosis complex DNA were employed, including analysis of paleontologic and modern specimens in 2 geographically separate laboratories.

PCR primers that can detect low levels of Mycobacterium leprae DNA.

There are several specific PCR-based methods to detect Mycobacterium leprae DNA, but the amplicons are quite large. For example, primers that target the 36-kDa antigen gene and are in common diagnostic use yield a 530-bp product. This may be a disadvantage when examining samples in which the DNA is likely to be damaged and fragmented. Therefore, two sets of M. leprae-specific nested primers were designed, based on existing primer pairs which have been shown to be specific for M. leprae. Primers that targeted the 18-kDa antigen gene gave an outer product of 136 bp and inner product of 110 bp. The primers based on the RLEP repetitive sequence yielded a 129-bp outer product and 99-bp nested product. With dilutions of a standard M. leprae killed whole-cell preparation as the source of DNA, both single-stage and nested PCR were performed after optimisation of the experimental conditions. Compared with the 36-kDa antigen gene primers, the 18-kDa antigen gene outer primers were 100-fold more sensitive and the RLEP outer primers were 1000-fold more sensitive. As an illustration of two possible applications of these new primers, positive results were obtained from three skin slit samples from treated lepromatous leprosy patients and three archaeological samples from human remains showing typical leprosy palaeopathology. It was concluded that these new primers are a useful means of detecting M. leprae DNA which is damaged or present at a very low level.

Tropheryma whippelii DNA in saliva of healthy people.

In a random sample of 40 healthy people, 35% showed evidence of Tropheryma whippelii DNA in their saliva. Consistent detection of T. whippelii DNA on repeated sampling suggests that this organism can be an oral commensal.

Mycobacterium tuberculosis complex DNA in calcified pleura from remains 1400 years old.

Mycobacterium tuberculosis complex DNA was isolated and identified in calcified pleura from remains 1400 years old, with the polymerase chain reaction. This is the first demonstration of tuberculosis in non-mummified archaeological tissue other than bone; the presence of mycobacterial mycolic acids in the sample supports this conclusion. The study of ancient DNA from microbial pathogens is of interest as it enables verification of traditional diagnoses, may answer long-standing questions in the history of disease, and provides ancient DNA sequences that can be compared with those of modern isolates.

The influence of live weight, live-weight change and diet on protein synthesis in the skin and skeletal muscle in young Merino sheep.

Wool growth is derived directly from protein synthesis in the skin of sheep, and is affected by the nutritional status of the animals. The present experiment examined both protein synthesis in the skin and muscle and wool growth in Merino lambs differing in live weight, intake and dietary protein source. The experiment was a 2(3) factorial design: twenty-four 5-month-old lambs initially weighing 33 kg (heavy) or 25 kg (light) were fed on a hay-based diet with either lupin seed or rapeseed meal as the major protein sources to maintain live weight (M) for 56 d, or were fed at 0.6 M for 28 d (period 1) followed by 28 d at 1.6 M (period 2). Fractional protein synthesis rates (FSR, % per d) in the skin and the m. longissimus dorsi on days 4 and 24 of period 1 and day 4 of period 2 were measured by means of a flooding dose of L-[ring-d5]phenylalanine, and wool growth on a skin patch over period 1 was also measured. The FSR ranged from 13.2 to 20.2% per d in the skin, higher than reported for other breeds, and 1.53-3.07% per d in the muscle. Sheep on the low intake (0.6 M) had significant reductions in FSR, protein content (g), protein synthesis (g/d) in the skin, and wool growth (g/d). The heavy lambs had similar FSR to the light lambs, but had a higher skin protein content and total protein synthesis per unit of skin area (100 cm2) and, therefore, grew more wool. The rapeseed-meal diet increased FSR and wool growth only in the light lambs over the short term. The protein deposited in wool over period 1 was 0.185 of the total protein synthesis in the skin, regardless of live weight, intake or diet, a result similar to other breeds. With the changes in dietary intake, protein synthesis in the skin and muscle responded differentially, with nutrient partitioning at sub-maintenance in favour of wool growth but at supra-maintenance, following a nutrient restriction, in favour of weight gain in young growing sheep.

A longitudinal study of environmental mycobacteria on a farm in south-west England.

Soil, stream beds and cattle drinking troughs were sampled every 3 months over 3 years. More than 750 putative mycobacteria were isolated and grouped into more than 50 biotypes pending full identification. Samples from woodland and farmed land yielded fewer isolates per site compared with other terrains (P < 0.05). Some seasonal effects were noted but the greatest difference was between years 1 and 3. This appeared not to be due to differences in temperature, rainfall or experimental procedure, but coincided with the introduction of organic farming practices. In year 3 there was a significant increase in nitrate-reducing slow growers, both pigmented (P < or = 0.006) and non-pigmented strains (P < or = 0.002), and a shift in biotypes was noted. In contrast, all fast growers declined with time, as did those slow growers unable to reduce nitrate. Changing farming practice may alter the profile of environmental mycobacteria, which has important implications for the immunological priming of humans and animals.

Variation within Mycobacterium scrofulaceum.

The purpose of this study was to identify Mycobacterium scrofulaceum reliably and rapidly and investigate diversity within the species. Fifty-four cultures were identified as Myco. scrofulaceum by preliminary cultural and biochemical tests, thin-layer chromatography and double diffusion. These strains were examined by PCR based on the 65 kDa heat stress protein gene, followed by restriction enzyme analysis of the product with BstEII and HaeIII. This produced seven groups, most with fewer fragments than had been reported previously. The technique was a rapid and reliable method for studying variation within Myco. scrofulaceum but alone, was unable to discriminate between some of these variants and other genetically similar species. When PCR-RFLP results were combined with biochemical tests, the major groups appeared to relate to different disease situations and thus, may have some epidemiological value.

Cyclosporine a immunosuppression in sheep with response enhancement by concomitant ketoconazole.

1. The pharmacokinetics of a single dose of Cyclosporine A (CsA) administered to sheep by intravenous (i.v.) route were examined. 2. Concomitant administration of ketoconazole was found to increase the area under the blood CsA concentration-time curve (AUC) and was effective when administered by the oral or intraperitoneal route. 3. The effects of CsA and ketoconazole on the immune system of sheep were also assessed. 4. A single dose of CsA 5 mg/kg resulted in abrogation of in vitro lymphocyte function manifest at 24 h after injection of CsA. Normal responsiveness recovered in 48-72 h. Numbers of T lymphocytes in peripheral blood were elevated transiently at 48 h although no other significant alteration in lymphocyte subsets was observed with this treatment. 5. Concomitant ketoconazole administration enhanced the CsA-induced suppression of in vitro lymphocyte responses. Blood levels of CsA (AUC values to 24 h) were significantly elevated with concomitant ketoconazole administration and depression of lymphocyte responses to mitogens were also significantly enhanced. An increase in the proportion of T4 positive cells in the blood was observed at 48 h and at 7 days after administration of CsA with ketoconazole. 6. These findings indicate that CsA effectively abrogates immunocompetence in the sheep and this immunosuppressive effect is enhanced by concomitant administration of ketoconazole.

Application of polymerase chain reaction for the detection of Mycobacterium leprae DNA in specimens from treated leprosy patients.

In this study of leprosy patients apparently cured by dapsone monotherapy, the polymerase chain reaction (PCR), one of the most reliable and sensitive DNA-based assays, was used for the specific detection of Mycobacterium leprae DNA. Sputum and slit-skin samples from 44 such patients at Baba Baghi Leprosy Sanatorium in Iran were examined. Primers for a 530-base-pair fragment of the gene encoding the 36-kDa antigen of M. leprae were used for the study. The PCR results were compared with microscopy for acid-fast bacilli. Of the 44 sputum samples, 2 were positive by PCR (4.5%) and of the 44 slit-skin swabs taken from the same patients, 10 were PCR positive (22.7%). Only one patient was PCR positive for both sputum and slit-skin specimens (2.3%). No positive results were found by acid-fast microscopy. In total, 11 of 44 (25%) patients in this study were found to be PCR positive for M. leprae, and it was thought probable that this indicated the presence of live organisms. Particularly interesting was the statistically significant association of positive results from slit-skin swabs with paucibacillary rather than multibacillary leprosy. It is suggested that whereas relapse or immunological reaction in paucibacillary disease may result from surviving organisms, in multibacillary leprosy this may be due to re-infection.

Radiopharmaceutical therapy of 5T33 murine myeloma by sequential treatment with samarium-153 ethylenediaminetetramethylene phosphonate, melphalan, and bone marrow transplantation.

BACKGROUND: Total-body irradiation, followed by hematopoietic system rescue by bone marrow transplantation (BMT), has been found to improve the response of patients with multiple myeloma to treatment with melphalan. The problems of nonhematopoietic toxicity from whole-body irradiation might be circumvented by using a bone-seeking radiopharmaceutical, such as samarium-153 ethylenediaminetetramethylene phosphonate (153Sm-EDTMP), to ablate the bone marrow. PURPOSE: A mouse model system for multiple myeloma was used to evaluate the potential therapeutic efficacy of sequential therapy with 153Sm-EDTMP, melphalan, and BMT. METHODS: Female C57BL/KaLwRij mice were inoculated with 8 x 10(5) 5T33 murine myeloma cells. Treatment protocols were begun 3 or 10 days later, when the myeloma was either confined to bone marrow or disseminated in liver, spleen, and lymph nodes, simulating human multiple myeloma. 153Sm, a potent beta particle-emitting radioisotope of short half-life (46.7 hours), was linked to the bone-seeking chelate EDTMP. Animals in the first treatment group were each given 22.5 MBq 153Sm-EDTMP via the jugular vein (day 3 or 10), followed by 18.5 mg/kg melphalan (maximum tolerated dose) given intraperitoneally 5 days later (day 8 or 15) and syngeneic BMT another 2 days later (day 10 or 17). Survival in groups of six to 10 animals for each time series was compared with that in mice left untreated (control cohort), in mice treated with 153Sm-EDTMP alone (day 3 or 10), or in mice treated with melphalan alone (day 8 or 15). The hematopoietic systems of animals in the latter two treatment groups recovered full function, obviating the necessity of BMT. The end point was onset of paraparesis, at which time the animals were immediately killed by carbon dioxide asphyxiation. RESULTS: Median survival in untreated control animals was 23 days in those with localized disease and 24 days in those with disseminated myeloma. Treatment with 153Sm-EDTMP alone improved survival to a median of 29 days when commenced on day 3 and 30 days when begun on day 10. Melphalan treatment alone improved the median survival to 31 days for animals with localized myeloma and 34 days in animals with disseminated disease. Additional improvement in survival to a median of 42 days was achieved in animals treated 3 days after tumor inoculation with sequential 153Sm-EDTMP, melphalan, and BMT; median survival was 40 days using this regimen in animals with disseminated myeloma. CONCLUSIONS: Animals in all three treatment protocols survived longer than those left untreated after inoculation with myeloma cells (P < .001). Sequential treatment with 153Sm-EDTMP, melphalan, and BMT was significantly more effective than single-agent treatment (P < .01). No evidence of radiotoxicity was detected in nonhematopoietic organs. IMPLICATIONS: The survival advantage conferred by our sequential treatment protocol suggests its potential clinical usefulness in the treatment of multiple myeloma and other hematologic malignancies in humans.

A model of multiple myeloma: culture of 5T33 murine myeloma cells and evaluation of tumorigenicity in the C57BL/KaLwRij mouse.

The 5T33 multiple myeloma is one of a series of transplantable murine myelomas arising spontaneously in C57BL/KaLwRij mice. This study describes the establishment and characterisation of the 5T33 murine myeloma in vitro as a cultured cell line in terms of its morphology, growth rate, expression of paraprotein (IgG2b) and tumorigenicity in syngeneic animals. The 5T33 cell line has been in continuous culture for over 10 months and has achieved more than passage 34. In culture, 5T33 myeloma grows as single cells or in small clusters of loosely adherent cells on an adherent stromal cell layer. Maximum doubling time is approximately 25 h, and over 90% of the cells express cytoplasmic IgG2b paraprotein. The cultured 5T33 myeloma cells are highly tumorigenic in C57BL/KaLwRij mice with as few as 500 cells inducing paralysis and death as early as day 36 post-tumour inoculation. Kinetics of tumour development and detection of IgG2b paraprotein are dose dependent. Two weeks following intravenous inoculation of 5 x 10(5) cultured 5T33 myeloma cells, tumour cells were readily identified in the bone marrow. By 3 weeks post-tumour inoculation, 5T33 myeloma cells were found in various tissues throughout the animal. Studies are now underway to determine the sensitivity of this cell line to various therapeutic modalities.