Therapeutic drug monitoring and TB treatment outcomes in patients with diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) increases the risk of TB disease and poor treatment outcomes such as delayed sputum culture conversion due to inadequate drug exposure. Therapeutic drug monitoring (TDM) has improved these outcomes in some settings.METHODS: To compare treatment outcomes in programs with routine TDM vs. programs that did not use TDM, we conducted a retrospective study among people with DM and TB at health departments in four US states.RESULTS: A total of 170 patients were enrolled (73 patients in the non-TDM group and 97 patients in the TDM group). Days to sputum culture conversion and total treatment duration were significantly shorter in the TDM group vs. the non-TDM group. In adjusted analyses, patients who underwent TDM were significantly more likely to achieve sputum culture conversion at 2 months (P = 0.007).CONCLUSION: TDM hastened microbiological cure from TB among people with DM and a high risk for poor treatment outcomes in the programmatic setting.

Non-adherence and drug-related interruptions are risk factors for delays in completion of treatment for tuberculosis.

SETTING: A key program performance objective established by the Centers for Disease Control and Prevention (CDC) is that ≥93% of tuberculosis (TB) cases complete treatment within 12 months. OBJECTIVE: To determine the rate of and risk factors for delay in anti-tuberculosis treatment completion. DESIGN: Nested case-control study among TB cases reported to the Tennessee Department of Health between 1 January 2000 and 31 December 2010. Time to complete treatment was calculated using treatment start and stop dates documented in the Tuberculosis Information Management System (TIMS). RESULTS: Of 2627 cases, 261 (9.9%) required >12 months to complete treatment. In adjusted conditional logistic regression analyses, cavitary disease and positive cultures after 2 months of therapy (OR 5.85, 95%CI 1.98-17.32, P = 0.001), non-adherence (OR 4.13, 95%CI 1.76-9.72, P < 0.001), and interruptions in treatment due to drug-related issues (OR 6.91, 95%CI 3.76-12.70, P < 0.001) were independently associated with delay in completion of TB treatment. CONCLUSION: From 2000 to 2010, the proportion of TB cases completing treatment within 12 months increased from 84.6% to 94.9%, and remained above the CDC target during 2009-2010. Efforts to improve patient adherence and reduce interruptions in treatment due to anti-tuberculosis drug-related issues could improve the proportion of TB cases completing treatment within 12 months.

Chronic lung disease and HIV infection are risk factors for recurrent tuberculosis in a low-incidence setting.

SETTING: Programmatic data from the United States on tuberculosis (TB) recurrence are limited. OBJECTIVES: To determine the TB recurrence rate and to determine if chronic lung disease (CLD) and human immunodeficiency virus (HIV) infection are risk factors for recurrence in this population. DESIGN: Nested case-control study among TB cases reported to the Tennessee Department of Health between 1 January 2000 and 31 December 2006. Time at risk for recurrence was through 31 December 2007. Multiple imputation accounted for missing data. RESULTS: Of 1431 TB cases, 20 cases recurred (1.4%, 95%CI 0.9-2.1). Median time at risk for recurrence was 4.5 years (interquartile range 2.7-6.1). Initial and recurrent Mycobacterium tuberculosis isolates were available for genotyping for 15 patients; 12 were consistent with relapse (0.8%, 95%CI 0.4-1.5) and three with re-infection (0.2%, 95%CI 0.04-0.6). HIV infection (OR 5.01, P = 0.04) and CLD (OR 5.28, P = 0.03) were independently associated with recurrent TB, after adjusting for a disease risk score. HIV infection was a risk factor for TB re-infection (P < 0.001). CONCLUSIONS: In this low-incidence US population, the TB recurrence rate was low, but CLD and HIV were independent risk factors for recurrence. HIV infection was also a risk factor for TB re-infection.

Dose-response studies for pituitary and testicular function in male dogs treated with the GnRH superagonist, deslorelin.

We tested the effect of dose of GnRH superagonist on pituitary and testicular function in a study with four groups of four male dogs. The Controls received blank implants and the other three groups received implants containing 3, 6 or 12 mg deslorelin (D-Trp6-Pro9-des-Gly10-GnRH ethylamide). In all deslorelin-treated groups, there was initially an acute increase in plasma concentrations of LH and testosterone, followed by declines such that both hormones became undetectable after approximately 12 days. There was a dose-response in some of these early aspects of the hormone profiles. With respect to long-term effects of treatment, the 12-mg dose had significantly greater effects than the smaller doses for the duration of minimum testicular volume [366 +/- 77, mean +/- SEM (3 mg), 472 +/- 74 (6 mg), and 634 +/- 59 (12 mg) days], absence of ejaculate [416 +/- 88 (3 mg), 476 +/- 83 (6 mg), and 644 +/- 67 (12 mg) days], undetectable plasma concentrations of LH and testosterone [367 +/- 64 (3 mg), 419 +/- 72 (6 mg), and 607 +/- 69 (12 mg) days], the delay until complete recovery of LH and testosterone secretion [394 +/- 65 (3 mg), 484 +/- 72 (6 mg) and 668 +/- 47 (12 mg) days], and the delay until testes had regrown to normal volume [408 +/- 77 (3 mg), 514 +/- 74 (6 mg), 676 +/- 59 (12 mg) days]. The time taken to restore full ejaculates was also longest for the 12-mg dose: 716 +/- 67 (12 mg) days vs 440 +/- 66 (3 mg) and 538 +/- 83 (6 mg) days after implantation. There was no correlation between delay to recovery of normal ejaculate quality and body mass. We conclude that the dose-response relationship with deslorelin implants is not expressed with respect to the degree of suppression of reproduction, but on the maximum duration of suppression and thus to delay until recovery.

Morphological study of the effects of the GnRH superagonist deslorelin on the canine testis and prostate gland.

The present study is part of a programme of research designed to evaluate the efficacy of the GnRH superagonist,deslorelin (D-Trp6-Pro9-des-Gly10-LHRH ethylamide), as a contraceptive for male dogs. Adult dogs were assigned to a completely randomized design comprising six groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other five groups received a 6 mg deslorelin implant. One group of deslorelin treated dogs was sacrificed on each of days 16, 26, 41, 101 and 620, and testicular and prostate tissues were collected for study by light and electron microscopy. On days 16 and 26 after implantation, we observed partial disruption of the seminiferous tubules, with early spermatids shed into the lumen. On days 41 and 101 after implantation, 90­100% of the seminiferous tubules were atrophic and aspermatogenic.On day 101 after implantation, 99% of all sections showed atrophy of the epithelium and shrinkage of epithelial height in the ductus epididymides. On days 41 and 101 after implantation, prostate tissue showed complete atrophy of the glandular epithelium (100% of sections) and an apparent increase in the relative proportion of connective tissue. At the electron microscopic level, in dogs treated with deslorelin for 41 and 101 days, the Sertoli cells were smaller and their nucleoli appeared smaller than in the control dogs. The nucleoli of the Leydig cells were atrophied and prostate glandular epithelium showed reduced epithelial height, a trophy of the nucleolus and an absence of secretory granules.Tissues collected during the recovery phase revealed a complete recovery of spermatogenesis. In conclusion, slow release implants containing deslorelin induce a striking a trophy of the testes and prostate gland by 26 days after implantation, explaining the previously reported loss of ejaculate and arrest of sperm output. At histological level,the entire process appears to be completely reversible, in accordance with data on endocrine variables and semen production.

Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.

In an attempt to determine whether muscle-derived stem cells are distinct from satellite cells, we investigated whether muscle-derived stem cells could be isolated from the skeletal muscle of Pax7-deficient mice, which have been shown to be devoid of or to contain only a minimal number of satellite cells. Utilizing a technique that separates cells based on their adhesion characteristics (the preplate technique), several distinct populations of muscle-derived cells were isolated. In these mice, the Pax7 gene was knocked out with the insertion of the LacZ gene. One population was both rapidly adhering, LacZ-positive, and displayed a high myogenic index, but was rapidly lost to terminal differentiation when continuously replated. A second population, which persisted over 50 passages, was LacZ-negative and displayed a low myogenic index. Although Pax3 may have acted as a compensatory mechanism for the myogenic commitment of the LacZ-positive cells, the LacZ-negative cells, despite expressing Pax3, required Pax7 transduction to restore their myogenic capacity. We believe that these two populations of myogenic progenitor cells, each endowed with different adhesion characteristics, may help explain the discrepancy in the literature concerning the presence of myogenic cells found in Pax7-deficient mice.

Pituitary and testicular endocrine responses to exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone in male dogs treated with GnRH agonist implants.

The present study tested whether exogenous gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) can stimulate LH and testosterone secretion in dogs chronically treated with a GnRH superagonist. Twenty male adult dogs were assigned to a completely randomised design comprising five groups of four animals. Each dog in the control group received a blank implant (placebo) and each dog in the other four groups received a 6-mg implant containing a slow-release formulation of deslorelin (d-Trp6-Pro9-des-Gly10-LH-releasing hormone ethylamide). The same four control dogs were used for all hormonal challenges, whereas a different deslorelin-implanted group was used for each challenge. Native GnRH (5 microg kg(-1) bodyweight, i.v.) was injected on Days 15, 25, 40 and 100 after implantation, whereas bovine LH (0.5 microg kg(-1) bodyweight, i.v.) was injected on Days 16, 26, 41 and 101. On all occasions after Day 25-26 postimplantation, exogenous GnRH and LH elicited higher plasma concentrations of LH and testosterone in control than deslorelin-treated animals (P < 0.05). It was concluded that, in male dogs, implantation of a GnRH superagonist desensitised the pituitary gonadotrophs to GnRH and also led to a desensitisation of the Leydig cells to LH. This explains, at least in part, the profound reduction in the production of androgen and spermatozoa in deslorelin-treated male dogs.

Mechanisms of sperm-egg interactions emerging from gene-manipulated animals.

Untangling the molecular nature of sperm-egg interactions is fundamental if we are to understand fertilization. These phenomena have been studied for many years using biochemical approaches such as antibodies and ligands that interact with sperm or with eggs and their vestments. However, when homologous genetic recombination techniques were applied, most of the phenotypic factors of the gene-manipulated animals believed "essential" for fertilization were found to be dispensable. Of course, all biological systems contain redundancies and compensatory mechanisms, but as a whole the old model of fertilization clearly requires significant modification. In this review, we use the results of gene manipulation experiments in animals to propose the basis for a new vision.

Implications of micro-RNA profiling for cancer diagnosis.

Micro-RNAs (miRNAs) are a large class of small non-coding RNAs that regulate protein expression in eucaryotic cells. Initially believed to be unique to the nematode Caenorhabditis elegans, miRNAs are now recognized to be important gene regulatory elements in multicellular organisms and have been implicated in a variety of disease processes, including cancer. Advances in expression technologies have facilitated the high-throughput analysis of small RNAs, identifying novel miRNAs and showing that these genes may be aberrantly expressed in various human tumors. These studies suggest that miRNA expression profiling can be correlated with disease pathogenesis and prognosis, and may ultimately be useful in the management of human cancer.

Regeneration of dystrophin-expressing myocytes in the mdx heart by skeletal muscle stem cells.

Cell transplantation holds promise as a potential treatment for cardiac dysfunction. Our group has isolated populations of murine skeletal muscle-derived stem cells (MDSCs) that exhibit stem cell-like properties. Here, we investigated the fate of MDSCs after transplantation into the hearts of dystrophin-deficient mdx mice, which model Duchenne muscular dystrophy (DMD). Transplanted MDSCs generated large grafts consisting primarily of numerous dystrophin-positive myocytes and, to a lesser degree, dystrophin-negative non-myocytes that expressed an endothelial phenotype. Most of the dystrophin-positive myocytes expressed a skeletal muscle phenotype and did not express a cardiac phenotype. However, some donor myocytes, located at the graft-host myocardium border, were observed to express cardiac-specific markers. More than half of these donor cells that exhibited a cardiac phenotype still maintained a skeletal muscle phenotype, demonstrating a hybrid state. Sex-mismatched donors and hosts revealed that many donor-derived cells that acquired a cardiac phenotype did so through fusion with host cardiomyocytes. Connexin43 gap junctions were not expressed by donor-derived myocytes in the graft. Scar tissue formation in the border region may inhibit the fusion and gap junction connections between donor and host cells. This study demonstrates that MDSC transplantation warrants further investigation as a potential therapy for cardiac dysfunction in DMD.

The role of mitochondria in the establishment of oocyte functional competence.

Mitochondria are maternally inherited, semi-autonomous organelles with their own genomes (mtDNA), largely responsible for the generation of energy in the form of cellular ATP. However, mitochondrial replication and transcription of mtDNA do not commence until well into embryonic differentiation. This means that the oocyte needs to contain sufficient stocks of functioning mitochondria to fuel the first few days of embryonic development. In this review, I examine how qualitative and quantitative aspects of mitochondria help us define the notion of functional competence.

Muscle cell-mediated gene delivery to the rotator cuff.

Rotator cuff lesions are one of the most common causes of upper extremity disability. Surgical therapy addresses mostly the extrinsic etiology, but not intrinsic factors such as aging, structural changes, low vascularity, and inflammatory processes. In this study, genetically engineered, highly purified muscle-derived cells (MDCs) were characterized and injected into the supraspinatus tendons of nude rats. The injected cells were monitored for 3 weeks. In vitro, the engineered, highly purified MDCs do not express vimentin; 98% of them are positive for the beta-galactosidase marker gene, and 99% hybridize with the specific pancentromeric mouse probe. beta-Galactosidase marker gene expression of the injected cells was detected up to 21 days. From day 7 after injection, the cell nuclei became spindle shaped, cells were integrated into the tendon collagen bundles, and the cells showed differentiation into vimentin-expressing fibroblastic cells. The results indicate that the rotator cuff tendon matrix and its original cellular components modulated the injected MDCs toward a fibroblastic phenotype. The compatibility and ability of MDCs to differentiate into other cell lineages, such as fibroblasts, might have high potential utility in tissue-engineering applications for tendon healing. This approach facilitates the application of muscle-derived progenitor cells and ex vivo gene therapy for the treatment of rotator cuff lesions.

Use of a new drug delivery formulation of the gonadotrophin-releasing hormone analogue Deslorelin for reversible long-term contraception in male dogs.

In the present study, we tested the effect of treatment with a slow-release implant containing the gonadotrophin-releasing hormone agonist Deslorelin(TM) (Peptech Animal Health Australia, North Ryde, NSW, Australia) on pituitary and testicular function in mature male dogs. Four dogs were treated with Deslorelin (6-mg implant) and four were used as controls (blank implant). In control dogs, there were no significant changes over the 12 months of the study in plasma concentrations of luteinising hormone (LH) or testosterone, or in testicular volume, semen output or semen quality. In Deslorelin-treated dogs, plasma concentrations of LH and testosterone were undetectable after 21 and 27 days, testicular volume fell to 35% of pretreatment values after 14 weeks and no ejaculates could be obtained after 6 weeks. Concentrations returned to the detectable range for testosterone after 44 weeks and for LH after 51 weeks and both were within the normal range after 52 weeks. Semen characteristics had recovered completely by 60 weeks after implantation. At this time, the testes and prostate glands were similar histologically to those of control dogs. We conclude that a single slow-release implant containing 6 mg Deslorelin has potential as a long-term, reversible antifertility agent for male dogs.

The role of maternal mitochondria during oogenesis, fertilization and embryogenesis.

This review examines the place of mitochondria in the life cycle through oogenesis, ovulation and early embryogenesis. Mitochondria are semi-autonomous organelles responsible for the bulk of oxidative energy production in the body. They play central roles in ageing, in apoptosis and in many non-Mendelian-inherited bioenergetic and neurological diseases. Originating as free alpha-proteobacteria that entered into a symbiotic relationship with the ancestral eukaryotic organisms, they now have a highly restricted genome of ~16 kb, encoding for 37 genes of the oxidative phosphorylation pathway. Mitochondria are inherited through the mother and special mechanisms have evolved to eliminate the contribution of the spermatozoon in early embryonic development. Most mitochondrial genes have become translocated to the nucleus, and nuclear and mitochondrial genes have co-evolved. This, coupled with a high mutation rate in the remaining mitochondrial DNA, has resulted in a high degree of concordance between them. Disharmony between nuclear and mitochondrial genes is thus likely to complicate cloning technology and the experimental reconstruction of chimeric embryos by cytoplasmic or nuclear transfer.

Mitochondrial DNA polymorphisms and fertility in beef cattle.

Four hundred and twenty-two beef cattle of two different breeds (purebred Hereford and composite multibreed) were characterized by polymerase chain reaction-restriction fragment length polymorphism, using the restriction enzymes ApaI, AvaII, HindIII, PstI, SpeI, SspI and TaqI in two regions (the D-loop and the ND-5 gene) of mitochondrial DNA. The association between molecular haplotypes and records on calving rate, defined as the mean number of live calves born per year over 4 years, were examined by analysis of variance. A significant association was found between calving rate and mitochondrial polymorphisms in both breeds. This may have implications for genetically improving cow fertility.

ASCOT: a comprehensive clinical database for gastro-oesophageal cancer surgery.

We report the development of a multicentre prospective database for gastro-oesophageal cancer surgery. The ASCOT project aims to collect detailed prospective data on co-morbidity, surgery, stage and outcome in a large cohort of patients from a wide variety of hospitals in the UK. We describe the rationale for the initiative, the process of defining the dataset, the database software and structure, the system for data recording and retrieval and the operating rules of the ASCOT co-operative group. Thirty Trusts are currently submitting data, and the first 1000 cases have now been entered. A first annual report has been produced, showing anonymized comparative figures for patient characteristics and outcomes of interest, and a second is due shortly. The collection of detailed comparable data, including co-morbidity evaluation, on a large scale is likely to prove valuable for unit specialist accreditation, surgeon re-validation, audit and research.

Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility.

Human mitochondrial DNA polymerase, encoded by POLG, contains a polyglutamine tract encoded by a CAG microsatellite repeat. Analysis of POLG genotypes in different populations identified an association between absence of the common, ten-repeat allele and male infertility typified by a range of sperm quality defects but excluding azoospermia.

Effect of bone morphogenetic protein-2-expressing muscle-derived cells on healing of critical-sized bone defects in mice.

BACKGROUND: Cells that express bone morphogenetic protein-2 (BMP-2) can now be prepared by transduction with adenovirus containing BMP-2 cDNA. Skeletal muscle tissue contains cells that differentiate into osteoblasts on stimulation with BMP-2. The objectives of this study were to prepare BMP-2-expressing muscle-derived cells by transduction of these cells with an adenovirus containing BMP-2 cDNA and to determine whether the BMP-2-expressing muscle-derived cells would elicit the healing of critical-sized bone defects in mice. METHODS: Primary cultures of muscle-derived cells from a normal male mouse were transduced with adenovirus encoding the recombinant human BMP-2 gene (adBMP-2). These cells (5 yen 10(5)) were implanted into a 5-mm-diameter critical-sized skull defect in female SCID (severe combined immunodeficiency strain) mice with use of a collagen sponge as a scaffold. Healing in the treatment and control groups was examined grossly and histologically at two and four weeks. Implanted cells were identified in vivo with use of the Y-chromosome-specific fluorescent in situ hybridization (FISH) technique, and their differentiation into osteogenic cells was demonstrated by osteocalcin immunohistochemistry. RESULTS: Skull defects treated with muscle cells that had been genetically engineered to express BMP-2 had >85% closure within two weeks and 95% to 100% closure within four weeks. Control groups in which the defect was not treated (group 1), treated with collagen only (group 2), or treated with collagen and muscle cells without adBMP-2 (group 3) showed at most 30% to 40% closure of the defect by four weeks, and the majority of the skull defects in those groups showed no healing. Analysis of injected cells in group 4, with the Y-chromosome-specific FISH technique showed that the majority of the transplanted cells were located on the surfaces of the newly formed bone, but a small fraction (approximately 5%) was identified within the osteocyte lacunae of the new bone. Implanted cells found in the new bone stained immunohistochemically for osteocalcin, indicating that they had differentiated in vivo into osteogenic cells. CONCLUSIONS: This study demonstrates that cells derived from muscle tissue that have been genetically engineered to express BMP-2 elicit the healing of critical-sized skull defects in mice. The cells derived from muscle tissue appear to enhance bone-healing by differentiating into osteoblasts in vivo. CLINICAL RELEVANCE: Ex vivo gene therapy with muscle-derived cells that have been genetically engineered to express BMP-2 may be used to treat nonhealing bone defects. In addition, muscle-derived cells appear to include stem cells, which are easily obtained with muscle biopsy and could be used in gene therapy to deliver BMP-2.