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The use of Raman spectroscopy to determine the radiation response of human glioma cells and how this response differs depending on the cell's position in the cell cycle.

Koala Retrovirus (KoRV) has been widely speculated to cause immune suppression in koalas (Phascolarctos cinereus) and to underlie the koala’s susceptibility to infectious disease, however evidence for immunomodulation is limited. The aim of this study is to determine whether immunophenotypic changes are associated with KoRV infection in free ranging Victorian koalas. qPCR was used to examine mRNA expression for Th1 (IFNγ), Th2-promoting (IL6, IL10) and Th17 (IL17A) cytokines, along with CD4 and CD8 in whole blood of koalas (n = 74) from Mt Eccles and Raymond Island in Victoria, Australia, with and without natural chlamydial infection. KoRV positive koalas had significantly lower levels of IL17A (p`0.023) and IFNγ (p = 0.044) gene expression along with a decreased CD4:CD8 gene expression ratio (p = 0.025) compared to negative koalas. No effect of chlamydial infection or combined effect of KoRV and chlamydial infection was detected in these populations. The decreased expression of IFNγ could make KoRV infected koalas more susceptible to persistent chlamydial infection, and a decrease in IL17A could make them more susceptible to gram negative bacterial, fungal and mycobacterial infection; but more tolerant of chlamydial infection.

Background Providing compensation for participants in clinical research is well established and while international guidelines exist, defining a context-specific and fair compensation for participants in low-resource settings is challenging due to ethical concerns and the lack of practical, national compensation and reimbursement frameworks. Methods We reviewed Oxford University Clinical Research Unit (OUCRU) internal reimbursement documentation over a 10-y period and conducted a scoping literature review to expand our knowledge of compensation and reimbursement practices including ethical concerns. We developed a preliminary reimbursement framework that was presented to community advisory boards (CAB) and clinical investigators to assess its applicability, fairness and transparency. Results The main topics discussed at the workshops centered on fairness and whether the reimbursements could be perceived as financial incentives. Other decisive factors in the decision-making process were altruism and the loss of caregivers’ earnings. Investigators raised the issue of additional burdens, whereas the CAB members were focused on non-monetary elements such as the healthcare quality the patients would receive. All elements discussed were reviewed and, where possible, incorporated into the final framework. Conclusion Our new reimbursement framework provides a consistent, fair and transparent decision-making process and will be implemented across all future OUCRU clinical research in Vietnam.

As a “young person” I am unsurprised at the high number of physicians using WhatsApp to communicate in the healthcare setting.1 Many, if not the majority, of other businesses around the world use WhatsApp or similar programmes to communicate with each other instantly. But they will not be as concerned with information governance and confidentiality as …

Although koala retrovirus (KoRV) is widely termed a pathogen, direct evidence for causation of disease impacts in koalas (Phascolarctos cinereus) remains elusive. Examination of the immune system of koalas could provide a sharper tool to investigate this but progress has been slow due to a paucity of immunological reagents in this species, and historical contradictions in research findings in this area. Our work using cross reactive antibodies to examine behaviour of resting and stimulated koala T cells (anti-human CD3); B cells (anti-human CD79b); MHCII (anti-human HLA-DP, DQ, DR) and interferon gamma (anti-bovine IFNg) by flow cytometry have revealed some features consistent with a skew to a Th2 (B cell) immune focus. Assessing the role of KoRV in immunomodulation in koalas clearly requires more in-depth research. We have used recent advances in genomics of other marsupials to develop tools necessary to assess KoRV's effects on koala immune function in free-ranging, captive and in-vitro systems.

No information is currently available on the influence of injectable second-line drugs on treatment outcomes of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) patients. To investigate this issue, a large series of MDR- and XDR-TB cases diagnosed in Estonia, Germany, Italy and the Russian Federation (Archangels Oblast) between 1999 and 2006 were analysed. All study sites performed drug susceptibility testing for first- and second-line anti-TB drugs, laboratory quality assurance and treatment delivery according to World Health Organization recommendations. Out of 4,583 culture-confirmed cases, 240 MDR- and 48 XDR-TB cases had a definitive outcome recorded (treatment success, death, failure). Among MDR- and XDR-TB cases, capreomycin resistance yielded a higher proportion of failure and death than capreomycin-susceptible cases. Resistance to capreomycin was independently associated with unfavourable outcome (logistic regression analysis: odds ratio 3.51). In the treatment of patients with multidrug-resistant and extensively drug-resistant tuberculosis, resistance to the injectable drug capreomycin was an independent predictor for therapy failure in this cohort. As Mycobacterium tuberculosis drug resistance is increasing worldwide, there is an urgent need for novel interventions in the fight against tuberculosis.

To the Editor: Drug-resistant tuberculosis (TB) has been identified as a major problem in the former Soviet Union, and was recently surveyed in the Aral Sea regions of Dashoguz (Turkmenistan) and Karakalpakstan (Uzbekistan) (1). However, few data are available for the Caucasian region and published reports have focused mainly on prisons (2,3). We report a drug resistance survey for first- and second-line anti-TB drugs conducted in Abkhazia, a Caucasian region of 8,600 km2 with approximately 250,000 inhabitants, at the western end of Georgia on the Black Sea. The collapse of the Soviet Union lead to disruption of TB control activities in all Eastern bloc regions (4). In Abkhazia, the shortage and poor quality of drugs, self-medication, and poor adherence to the therapy became even more evident during the war with Georgia in 1993 and the international embargo that followed. A TB program based on the World Health Organization/International Union against Tuberculosis and Lung Disease (WHO/IUATLD) recommendations was initiated in Abkhazia with the support of Medecins Sans Frontieres (MSF) in 1999. In 2000, monitoring of drug resistance was started for new cases and previously treated case-patients. The study was performed in collaboration with the Guliripchi TB Hospital, MSF, and the Istituto Superiore di Sanita (ISS), a WHO/IUATLD Supranational Reference Laboratory for anti-TB drug resistance. Sputa were collected from all patients attending Guliripchi TB Hospital in Sukhumi, the capital of Abkhazia, from September 2000 to April 2004. Patients were either referred by their practitioners or came spontaneously because TB was suspected. Diagnosis, treatment, and hospitalization were provided free. Samples were treated as previously described (5). Of 489 sputa collected from individual patients, 447 were culture positive (246 from new case-patients and 201 from previously treated case-patients) and 42 were culture negative; of these, >90% showed a negative, doubtful, or 1+ smear result. Susceptibility to first-line (streptomycin, isoniazid, rifampin, and ethambutol) and second-line (kanamycin, ethionamide, capreomycin, cycloserine, p-aminosalicylic acid, and ofloxacin) drugs was determined by the proportion method on Middlebrook 7H10 agar. The critical concentrations used were streptomycin, 2 µg/mL; isoniazid, 0.2 µg/mL; rifampin, 1 µg/mL; ethambutol, 5 µg/mL; kanamycin, 5 µg/mL; ethionamide, 5 µg/mL; capreomycin, 10 µg/mL; p-aminosalicylic acid, 2 µg/mL; and ofloxacin, 2 µg/mL (6–8). Cycloserine was used at a concentration of 30 µg/mL (9). If a strain was resistant to >1 first-line drugs, the susceptibility to all second-line drugs was determined. Data on resistance to the first- and second-line drugs are given in the Table. The strains isolated from 35.8% of the new cases and 57.2% of the previously treated case-patients were resistant to >1 first-line drugs. The highest monoresistance was seen for isoniazid and streptomycin in both new and previously treated case-patients, while monoresistance to rifampin and ethambutol was low ( 1 second-line drugs. Table First-line and second line antituberculosis drug resistance in 447 Mycobacterium tuberculosis strains collected in Abkhazia from September 2000 to April 2004* Few data have been reported on drug resistance to first- and second-line drugs in the former Soviet Union and in the Caucasian region (1–4). Overall, in Abkhazia, monoresistance to isoniazid was higher than in Karakalpakstan and Dashoguz (1), while monoresistance to streptomycin was lower. MDR-TB in new and previously treated case-patients showed levels intermediate between these 2 regions. Resistance to kanamycin and ethionamide was 14.3% and 12.8%, respectively, while resistance to ofloxacin was low (1.5%). Fluoroquinolones have not been commonly used in Abkhazia and former regions of the Soviet Union. Currently, regimens for the treatment of MDR-TB in Abkhazia combine an intensive phase for a minimum of 6 months with at least 4 drugs to which the MTB strain is susceptible, including 1 parenteral agent and 1 fluoroquinolone (ofloxacin), followed by a continuation phase of at least 15 months with >3 drugs. This is the first survey reporting drug susceptibility data for MTB within the Caucasus. It indicates that the prevalence of MDR strains is similar to that in other central Asia regions (1). Our results are representative of the present situation in Abkhazia since sampling systematically covered all TB cases for the period examined. The Guliripchi TB Hospital of Sukhumi is the only TB treatment center in the region, and all cases were included in the study. Overall, our data show that second-line drug resistance is present in Abkhazia, particularly among cases with MDR, and suggest the adoption of strategies for access and correct use of second-line drugs (10).

Background Elevated temperatures jeopardize plant disease resistance, as mediated by salicylic acid (SA). SA potentiates heat-induced expression of the 70-kDa heat shock protein (Hsp70) in tomato cells. In mammalian cells, Hsp70 suppresses apoptosis. We hypothesized that potentiation of heat-induced Hsp70 by SA contributes to a reduction in apoptosis in tobacco protoplasts. Methods Tobacco protoplasts (Nicotiana tabacum) were exposed to SA (70 μM) at normal temperatures or in combination with heat shock. Hsp70/Hsc70 accumulation and phosphatidylserine (PS) exposure, DNA fragmentation, as well as loss of mitochondrial membrane potential were quantified by flow cytometry. Results and Conclusions SA at normal temperatures did not influence Hsp70/Hsc70 accumulation, but were found to induce apoptosis. In contrast, SA in combination with HS potentiated heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts that correlated negatively with apoptosis, illustrated by decreased PS exposure and DNA fragmentation and enhanced mitochondrial membrane potential. We propose that this correlation supports a possible role for apoptosis suppression by Hsp70 under elevated temperatures during pathogen infection. © 2004 Wiley-Liss, Inc.

The clinical antitumor efficacy of nitrogen mustards such as chlorambucil may relate to their ability to cause programmed cell death (apoptosis), probably through their DNA cross-linking properties. In contrast, bisbenzimidazoles such as Hoechst 33342 interact noncovalently with the minor groove of DNA, and appear to cause apoptosis in a fundamentally different way, which may involve the inhibition of topoisomerase (topo) I enzymes. A series of DNA minor groove binding nitrogen mustards with selective DNA affinity and in vivo antitumor activity in animal models was studied. Although two examples of such compounds proved to inhibit topo I enzymes in vitro, they were equally toxic towards topo I-proficient and- deficient strains of yeast, suggesting that topo I inhibition was not involved in cell killing. Flow cytometric analysis of Chinese hamster cells highlighted the differences in the propensity to cause apoptosis by chlorambucil compared with Hoechst 33342, revealing two distinct apoptotic populations in cells treated with the latter drug. Unexpectedly, the bisbenzimidazole mustards showed a novel peak of apoptotic activity, distinct from that shown by either parent drug. Exploring these different mechanisms of apoptosis may provide new directions for the development of antitumor drugs.

In 1998 a network of 20 regional tuberculosis (TB) laboratories (the Italian Multicentre Study on Resistance to Antituberculosis drugs (SMIRA) network) was established in Italy to implement proficiency testing and to monitor the prevalence of drug resistance nationwide. The network managed 30% of all TB cases reported in Italy each year. The aim of the present report is to describe: 1) the accuracy of drug-susceptibility testing in the network; 2) the prevalence of drug resistance for the period 1998-2000. Data were collected from the network laboratories. Sensitivity to streptomycin and ethambutol increased from the first survey (1998-1999) to the second survey (2000) from 87.7 to 91.9%. Specificity, predictive values for resistance and susceptibility, efficiency and reproducibility were consistent in both surveys. In previously untreated cases, the prevalence of multidrug-resistance was the same in both surveys (1.2%), while a slight decrease from the first to the second survey was observed for monoresistance to rifampicin (from 0.8 to 0.4%) and isoniazid (from 2.9 to 2%). The significant association found between isoniazid resistance and immigration is a useful indicator for both clinicians managing individual tuberculosis cases and public health services planning control strategies.

Death of suspension-cultured apple fruit cells ( Malus domestica Borkh. cv. Braeburn), resulting from exposure to low temperature (1 ° C), was reduced by a prior 1 h/38 ° C treatment. Pre-heated chilled cells produced less ethylene, indicating lower chilling injury. Heat treatment resulted in increased transcripts for a low molecular weight (hsp17) and a 70 kDa heat shock protein (hsp), but expression weakened within 24 h following the transfer of cells to 25 ° C. When cells were instead transferred to 1 ° ° ° C after heat, elevated hsp gene expression persisted for up to 3 d. Flow cytometric analysis of apple cell protoplasts showed that the 1 ° C treatment resulted in a subpopulation of protoplasts undergoing cell death, not observed with the 38 ° C pre-treatment. Chromatin condensation did not occur with the heat treatments. These results suggest that heatinduced reduction of cell death at low temperatures may involve persistence of hsps, and low-temperature susceptibility is less likely to be associated with programmed cell death. All cells do not respond in the same way, their tolerance being associated with different states of metabolism or of the cell cycle.

Tomato (Lycopersicon esculentum Mill.) is an important fruit crop world-wide and a model for studying fruit development. As determined using flow cytometry, fruit growth was characterized by high cell division activity in tomato during the first week after anthesis and followed by endoreduplications (DNA replication without cell divisions). D-type cyclins are considered to be important parts of the signal transduction for stimulation of DNA replication and cell division. To study the function of D cyclins in fruit development, full-length cDNA clones for three D cyclin genes were isolated from young tomato fruit. They were classified as D3 cyclins by sequence similarities and a phylogenetic analysis and named as LeCycD3;1, LeCycD3;2 and LeCycD3;3. The deduced amino acid sequences for LeCycD3;1-3 contained a retinoblastoma-binding motif and a PEST-destruction motif. Pollination and fertilization were followed by a high increase in the transcript levels of LeCycD3;1-3 in young fruit. Using in situ hybridization, high expression of LeCycD3;3 was detected in the vascular tissue of young fruit suggesting a role in vascular development. The D3 cyclins are probably involved in transducing the signals leading to fruit growth by cell divisions. Distinct differences were detected in their temporal and spatial expression patterns suggesting that they play different roles in fruit development as well as in the development of other plant organs.

The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) strains, i.e. strains resistant at least to isoniazid (INH) and rifampin (RMP), the two most powerful first-line drugs for the treatment of tuberculosis (TB), represents a worldwide health care problem. Patients with MDR strains should receive therapy based on individual drug susceptibility testing (DST) 1 including residual first-line (streptomycin, SM; ethambutol, EMB; and pyrazinamide, PZA) and second-line (ofloxacin, kanamycin, capreomycin, ethionamide, p-aminosalicylic acid, cycloserine) anti-TB drugs.

A large decrease in fluorescence intensity of propidium iodide (PI)-stained nuclei is observed during senescence of plant cells. The phenomenon reflects a decrease in accessibility of DNA to this fluorochrome and is a consequence of chromatin condensation. This decrease is substantially greater than usually found in animal nuclei whose chromatin undergoes condensation, e.g., during differentiation or quiescence. Chromatin condensation was confirmed by analyses of (i) DNA accessibility to DNase I, (ii) histone disassociation induced by HCl, (iii) saturation of binding sites by the PI fluorochrome (iv), and (v) visual inspection by fluorescence and confocal microscopy. The extent of changes revealed by these assays was used to map progressive changes in chromatin condensation which allowed us to identify different stages in an apoptosis-like pathway in plants. The initial step of chromatin condensation which occurred prior to endonucleolytic DNA degradation was detected by fluorescence and confocal microscopy and confirmed by a variety of assays employing flow cytometry. The initial chromatin condensation appears to be a reversible step in the early stage of apoptosis. The loss of reversibility of chromatin condensation observed subsequently may be a critical point in the cascade of apoptotic events, leading to further irreversible changes during apoptosis in plants.

Xyloglucan endotransglycosylase (XET) activity was measured in apple (Malus domestica Borkh. cv. Braeburn) pericarp and kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) outer pericarp and core tissues in order to establish whether a correlation exists between the activity of the enzyme and different stages of fruit development. Whereas the growth rate of kiwifruit paralleled changes in XET activity throughout fruit growth, that of apple did not. Both fruits showed the highest XET activity, on a fresh weight basis, in the first two weeks after anthesis when cell division was at its highest. XET activity then decreased sharply, but as the fruit increased in size (4-8 weeks after anthesis) there was a concomitant increase in XET activity in both fruits. In the latter stage of fruit development (16-26 weeks after anthesis) XET activity increased to peak at harvest in apple fruit. During this time there was relatively little increase in fruit size and presumably therefore minimal cell expansion. XET activity then declined as fruit softened after harvest. In core tissue from kiwifruit, XET activity increased throughout the later stages of fruit growth to harvest maturity in a similar manner to apple, but continued to increase after harvest until fruit were ripe. In contrast, XET activity in the outer pericarp of kiwifruit did not increase until ripening after harvest. In apple tissue up to 30% of the XET activity was cell wall bound and could not be solubilised, even in buffer containing 2 M NaCl. The results implicate XET in cell wall assembly during cell division and expansion early in apple and kiwifruit growth. However, the disparity between apple and kiwifruit with respect to XET activity late in fruit development and ripening and the different affinities of the enzyme for the cell wall in each fruit, suggest that XET has several roles in plant development, not all of which are related to cell wall loosening during periods of accelerated growth.

The establishment of the health-promoting benefits of probiotics is challenged by the antimicrobial bio-barriers throughout the host's gastrointestinal (GI) tract after oral administration. Although microencapsulation has been frequently utilised to enhance the delivery of probiotics, microcapsules of sub-100 μm were found to be ineffective and therefore questioned as an effective delivery vehicle for viable probiotics despite the sensory advantage. In this study, four probiotics strains were encapsulated in chitosan-coated alginate microcapsules of sub-100 μm. Only a minor protective effect was observed from this original type of microcapsule. In order to enhance the survival of these probiotics, sucrose, a metabolisable sugar, and lecithin vesicles were added to the wall material. Both of the ingredients could be readily encapsulated with the probiotics, and protected them from stresses in the simulated GI fluids. The metabolisable sugar effectively increased the survival of the probiotics in gastric acid, mainly through energizing the membrane-bound F1F0-ATPases. The lecithin vesicles proved to alleviate the bile salt stress, and hence notably reduced the viability loss at the elevated bile salt concentrations. Overall, three out of the total four probiotics in the reinforced sub-100 μm microencapsules could significantly survive through an 8-h sequential treatment of the simulated GI fluids, giving less than 1-log drop in viable count. The most vulnerable strain of bifidobacteria also yielded a viability increase of 3-logs from this protection. In conclusion, the sub-100 μm microcapsules can be a useful vehicle for the delivery of probiotics, as long as suitable protectants are incorporated in the wall matrix. Refereed/Peer-reviewed

Case-control study.The aim of this study was to assess the levels of psychological distress in patients with back pain who expressed claustrophobia at the time of their magnetic resonance imaging (MRI) scan, compared with sex and age-matched normal controls who did not exhibit claustrophobia. The secondary aim was to document the level of disability and intervention rates in this group.Psychosocial factors influence the outcomes of low back pain treatment with psychological distress being associated with poorer surgical outcomes in patients with low back pain. Up to 14% of patients experience claustrophobia during MRI scans requiring sedation to complete the scan. The effect of claustrophobia on back pain disability and outcomes has not been previously reported.Twenty females and 13 males all requiring MRI scan under sedation for claustrophobia (group 1) were compared with an age and sex-matched cohort that had MRI scan without sedation (group 2). Both groups were drawn from a chronic back clinic. Average age in both groups was 54 years (range, 27 to 79 y). Both groups had standard conservative therapy, together with psychometric evaluation: Zung Depression Index and Modified Somatic Perception Questionnaire. Disability was measured by Oswestry Disability Index. Primary outcome measures were intervention rates (surgery, injections, and physiotherapy sessions) and prevalence of psychological distress.Mean Zung Depression Index in group 1 was significantly higher than in group 2 (59.5 vs. 28.9, P0.05) as was the mean Modified Somatic Perception Questionnaire score (13.3 vs. 9.2, P0.05.) Prevalence of psychological distress was higher in group 1 (75.8% vs. 18.2%, P0.05). Oswestry Disability Index was the same in both groups (50% vs. 48%). Group 1 had 13 interventions (13 patients=39.4%) compared with 26 in group 2 (26 patients=78.8%) (P0.05). Twenty-two patients (66.7%) in group 1 were discharged after their MRI scan with no intervention compared with 7 patients (21.2%) in group 2 (P0.05).Claustrophobic patients with back pain showed higher levels of depression than nonclaustrophobic patients, with a higher rate of psychological distress. Disability, however, was not higher. The majority of claustrophobic patients did not require surgical intervention. The reasons for this are unclear and require further investigation. Claustrophobia requiring sedation for MRI scans may be a proxy for psychological distress in these patients and psychometric testing is advised during assessment to help with surgical decision making.

To assess prospectively the emotional content of dreams in individuals with the obstructive sleep apnea hypopnea syndrome (OSAHS) and sleepy snorers.Prospective observational study. Forty-seven patients with sleepiness and snoring attending a sleep-disordered breathing clinic, completed a morning diary concerning pleasantness/unpleasantness of their dreams for 10 days, and then had AHI assessed by a limited-channel home sleep study. Participants and groups: Sleepy snorers, AHI5: n = 12 (mean age = 51.00 years [SD 7.01], 7 males); AHI 5 -14.9, n = 14 (mean age = 49.71 y [9.73], 12 males); AHI ≥ 15, n = 21 (mean age = 56.33 [11.24], 16 males).All groups reported similar numbers of dreams and nightmares during the diary period. The AHI ≥ 15 group were significantly higher on dream unpleasantness than were the sleepy snorers (p0.05); and when only males were analyzed, this difference was also significant (p = 0.01). As AHI increased across the 3 groups, there was a significant decrease in variability of dream emotions (Levene test for homogeneity of variance between the 3 groups, p = 0.018). Mean daytime anxiety and daytime depression were significantly correlated with mean dream unpleasantness and with mean number of nightmares over the diary period.Patients with AHI ≥ 15 had more emotionally negative dreams than patients with AHI5. The variation in mean dream emotion decreased with increasing AHI, possibly because sleep fragmentation with increasing AHI results in fewer and shorter dreams, in which emotions are rarer.

The effect of 24- and 48-hour storage at room temperature on automated total nucleated cell count (TNCC), differential cell count (DCC) and cell morphology was assessed, and the effect of initial total protein concentration on canine and feline body cavity effusion samples (2 to 5 ml) was evaluated. At 24 and 48 hours, TNCC and absolute numbers of neutrophils, macrophages and small lymphocytes were significantly decreased and numbers of unrecognisable cells were significantly increased. Neoplastic cells and intracellular bacteria identified in fresh samples were missed at 24 and 48 hours. The initial total protein concentration was associated with an effect on percentage of unrecognisable cells and small lymphocytes over time. Change in TNCC over time would have resulted in misclassification of the effusion type in four of 47 samples.

The aim of this study was to use real-time polymerase chain reaction assays to determine the prevalence of three haemoplasma species in cats from Greece and to evaluate possible associations between haemoplasma infection and age, gender, feline immunodeficiency virus/feline leukaemia virus (FIV/FeLV) status and packed cell volume (PCV). Ninety-seven cats (24 ill anaemic, 55 ill non-anaemic, 18 healthy non-anaemic) were included in the study. Twenty cats (20.6%) were haemoplasma positive; seven cats were infected only with Mycoplasma haemofelis, 10 were infected only with ‘ Candidatus Mycoplasma haemominutum’ and three were co-infected with M haemofelis and ‘ Candidatus M haemominutum’. ‘ Candidatus Mycoplasma turicensis’ was not detected. Haemoplasma infection was associated with older age ( P=0.019). M haemofelis infection tended to be more common in anaemic cats ( P=0.058). No association between gender and haemoplasma infection, or haemoplasma relative copy number and PCV, was detected. Retroviral infection rates were very low with only one FeLV proviral positive cat found.

To the Editor: Twenty-three countries have reported >1 case of extensively drug-resistant tuberculosis (XDR TB) (1); however, information about XDR TB is still incomplete. In particular, the response of XDR TB to treatment in countries with low incidence is not known. We compared mortality rates from XDR TB with those from multidrug-resistant (MDR) TB. We analyzed data from all culture-confirmed TB cases diagnosed during 2003–2006 by the TB clinical reference centers in Italy (Sondalo, Milan, Rome) and Germany (Borstel, Grosshansdorf, Bad-Lippspringe) and reviewed original clinical records. Drug susceptibility testing for first- and second-line anti-TB drugs was performed according to World Health Organization (WHO) recommendations by quality-assured laboratories and retested at WHO Supranational Reference Laboratories (Rome/Milan; Borstel) (2–4). XDR TB was defined as resistance to at least rifampin and isoniazid (MDR TB definition) in addition to any fluoroquinolone and >1 of 3 injectable anti-TB drugs (capreomycin, kanamycin, amikacin) (3). Characteristics of MDR TB and XDR TB cases were compared by χ2 test (categorical variables), Student t test (admission days), and Kaplan-Maier curve (sputum smear, culture conversion), where appropriate. Of 2,888 culture-positive TB cases analyzed (Italy 2,140, Germany 748), 126 (4.4%) were MDR (Italy 83, Germany 43) and 11 (0.4%) were XDR (Italy 8, Germany 3). We estimate that the TB cases analyzed represent 24% of culture-positive cases reported in Italy (69.7% of MDR) and 4.2% of those reported in Germany (12.6% of MDR). XDR TB was diagnosed in each year of the study. All 11 XDR TB patients were receiving retreatment, and of the 126 MDR TB patients, 74 (58.7%) were receiving retreatment. All XDR TB patients were HIV seronegative; and of 109 MDR TB patients tested for HIV, 10 (9.2%) were HIV seropositive. Details about previous treatment regimens, drug resistance, and duration of treatment of XDR TB patients are summarized in the Appendix Table. XDR TB patients were significantly more likely than MDR TB patients to be resistant to all first-line drugs (8/11 vs. 36/126, p

Currently, no information is available on the effect of resistance/susceptibility to first-line drugs different from isoniazid and rifampicin in determining the outcome of extensively drug-resistant tuberculosis (XDR-TB) patients, and whether being XDR-TB is a more accurate indicator of poor clinical outcome than being resistant to all first-line anti-tuberculosis (TB) drugs. To investigate this issue, a large series of multidrug-resistant TB (MDR-TB) and XDR-TB cases diagnosed in Estonia, Germany, Italy and the Russian Federation during the period 1999-2006 were analysed. Drug-susceptibility testing for first- and second-line anti-TB drugs, quality assurance and treatment delivery was performed according to World Health Organization recommendations in all study sites. Out of 4,583 culture-positive TB cases analysed, 361 (7.9%) were MDR and 64 (1.4%) were XDR. XDR-TB cases had a relative risk (RR) of 1.58 to have an unfavourable outcome compared with MDR-TB cases resistant to all first-line drugs (isoniazid, rifampicin ethambutol, streptomycin and, when tested, pyrazinamide), and an RR of 2.61 compared with "other" MDR-TB cases (those susceptible to at least one first-line anti-TB drug among ethambutol, pyrazinamide and streptomycin, regardless of resistance to the second-line drugs not defining XDR-TB). The emergence of extensively drug-resistant tuberculosis confirms that problems in tuberculosis management are still present in Europe. While waiting for new tools which will facilitate management of extensively drug-resistant tuberculosis, accessibility to quality diagnostic and treatment services should be urgently ensured and adequate public health policies should be rapidly implemented to prevent further development of drug resistance.

Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biomet abolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformal dehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve TM , followed by permeabilization with Triton X-100 (0. 1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by;R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1. 2-fold increase in Hsp70/Hsc70 accumulation (P < 0. 001) in protoplasts, while Western blotting, quantified by image anal ysis, showed induction under similar conditions but at lower significance (P < 0. 05). The coefficients of variance for flow cytometry and Western blotting were 30. 7 and 49. 8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 °C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.

Programmed cell death (PCD) is an active way for plant cells marching to death, which plays an important role in plant development and stress responses. Cytological, biochemical, molecular and physiological methods for measuring plant PCD were reviewed. Application of flow cytometer to plant PCD research was also covered.