Low oral BCG doses fail to protect cattle against an experimental challenge with Mycobacterium bovis

Studies were undertaken to determine whether a dose of oral Mycobacterium bovis bacillus Calmette–Guérin (BCG) which did not induce skin test reactivity could protect cattle against bovine tuberculosis (TB). Groups of calves (n = 9) were vaccinated by administering 10⁸, 10⁷ or 10⁶ colony forming units (CFU) of BCG orally or 10⁶ CFU subcutaneous (s.c.) BCG. A control group (n = 10) was not vaccinated. All animals were challenged with M. bovis 18 weeks after vaccination and euthanized and necropsied at 16 weeks following challenge. Positive responses in the single cervical tuberculin skin test (severe interpretation) at 15 weeks post-vaccination were only observed in the s.c. BCG and 10⁸ CFU oral BCG groups (four of nine animals/group). Following experimental challenge with M. bovis, both these BCG-vaccinated groups had significant reductions in lesion scores and bacterial counts whereas there was no protection in calves vaccinated with oral doses of 10⁶ or 10⁷ CFU of BCG. In conclusion, low oral doses of BCG did not induce skin test responses, IFN-γ responses or protection against TB, however, in the BCG vaccine groups where protection was observed, there was no correlation between protection and skin test responses or IFN-γ responses.     

Vaccination of cattle with Mycobacterium bovis BCG by a combination of systemic and oral routes

Mycobacterium bovis bacille Calmette–Guérin (BCG) vaccine delivered to calves by the subcutaneous (s.c.) or by the oral route in a formulated lipid matrix has been previously shown to induce similar levels of protection against bovine tuberculosis. The current study was aimed at determining whether a combination of delivering BCG by s.c. and oral routes would enhance levels of protection, compared to only one route of vaccination. Forty calves were randomly divided into four groups (10/group). Calves were vaccinated with 10⁶ colony forming units (CFU) of BCG Pasteur by the s.c. route or orally with 10⁹ CFU BCG incorporated into a lipid formulation. One group received a combination of BCG administered by both the s.c. and oral routes and a non-vaccinated group served as a control. The two groups of calves that received s.c. BCG produced strong IFN-γ responses in whole blood cultures stimulated with bovine purified protein derivative (PPD) 3 weeks after vaccination. Cattle vaccinated just with oral BCG in a lipid matrix produced a strong IFN-γ response 8 weeks after vaccination, and peaking at 11 weeks after vaccination. All calves were challenged by the intratracheal route with M. bovis 15 weeks after vaccination and were euthanized and necropsied to assess protection at 17 weeks following challenge. BCG given s.c. or orally induced significant and comparable levels of protection against the virulent challenge. Vaccination of cattle by a combination of s.c./oral routes did not enhance protection beyond that achieved by s.c. or oral vaccination alone. We conclude that vaccination of cattle with BCG by a combination of routes has no beneficial additive effects, compared to a single s.c. administration of BCG or BCG given orally in a lipid formulation.     

Badger responses to small-scale culling may compromise targeted control of bovine tuberculosis

Where wildlife disease requires management, culling is frequently considered but not always effective. In the British Isles, control of cattle tuberculosis (TB) is hindered by infection in wild badger (Meles meles) populations. Large-scale badger culling can reduce the incidence of confirmed cattle TB, but these benefits are undermined by culling-induced changes in badger behavior (termed perturbation), which can increase transmission among badgers and from badgers to cattle. Test–vaccinate/remove (TVR) is a novel approach that entails testing individual badgers for infection, vaccinating test-negative animals, and killing test-positive animals. Imperfect capture success, diagnostic sensitivity, and vaccine effectiveness mean that TVR would be expected to leave some infected and some susceptible badgers in the population. Existing simulation models predict that TVR could reduce cattle TB if such small-scale culling causes no perturbation, but could increase cattle TB if considerable perturbation occurs. Using data from a long-term study, we show that past small-scale culling was significantly associated with four metrics of perturbation in badgers: expanded ranging, more frequent immigration, lower genetic relatedness, and elevated prevalence of Mycobacterium bovis, the causative agent of TB. Though we could not reject the hypothesis that culling up to three badgers per social group might avoid perturbation, we also could not reject the hypothesis that killing a single badger prompted detectable perturbation. When considered alongside existing model predictions, our findings suggest that implementation of TVR, scheduled for 2014, risks exacerbating the TB problem rather than controlling it. Ongoing illegal badger culling is likewise expected to increase cattle TB risks.Infectious diseases are often difficult to control where wildlife hosts contribute to pathogen persistence. Wildlife culling is a frequently considered control option, which is sometimes effective (1, 2), but often ineffective (3–6).In the United Kingdom, the cattle farming industry is seriously affected by bovine tuberculosis (TB) caused by Mycobacterium bovis (7). Selective culling of test-positive cattle has helped to eradicate TB across much of the developed world, but eradication from the United Kingdom is impeded by M. bovis infection in European badgers (Meles meles) (8), as well as by continued transmission among cattle (9–11). Transmission has also been documented among badgers (12), from cattle to badgers (13), and from badgers to cattle (14, 15). Because badgers are clearly a contributing factor to the UK’s TB problem, successive TB control policies have included culling of badgers (7, 8). To date, cattle controls have emphasized selective slaughter of test-positive animals, whereas badger culls have typically been nonselective, with no testing of live animals before culling (but see ref. 16).The impacts of nonselective badger culling on M. bovis transmission are well established. Such culling reduces badger density (17), but also promotes dispersal into the culled area (18) as well as expanding badger ranging in and around the areas where culls occurred (19). In Britain these behavioral changes—termed social perturbation—have been linked to increases in the proportion of badgers infected with M. bovis (13, 20), and reductions in the spatial clustering of infection (21). In cattle, the incidence of confirmed TB was reduced inside large culling areas where badger numbers were substantially suppressed by annual “proactive” culling. However, on adjoining unculled lands, and in areas receiving localized “reactive” culling, reductions in badger numbers were smaller, the incidence of confirmed cattle TB was elevated (14, 15, 22–24), and spatial clustering of cattle infection was reduced (21).This propensity of nonselective badger culling to prompt social perturbation and hence increase disease transmission is a major constraint on its utility as a tool for controlling cattle TB. An alternative approach, first proposed in the 1980s, would be to target culling at test-positive badgers, just as current controls target test-positive cattle (16, 25). A further elaboration, termed test–vaccinate/remove (TVR), involves killing test-positive badgers while vaccinating test-negative badgers. A pilot TVR program is scheduled to take place across 100 sq km in Northern Ireland in 2014 (26).Selective culling approaches (such as TVR) are likely to remove relatively small numbers of badgers. First, constraints on capture success limit testing to 56–85% of the badger population (27, 28). Second, not all captured badgers will be infected with M. bovis: in the 10 initial proactive culls of the Randomized Badger Culling Trial (RBCT), 2–38% of badgers had infection detectable by bacterial culture at standard necropsy (29). Third, not all infected badgers are detectable by available live tests: the only available trap-side test detected 49% of badgers that were culture-positive at necropsy (30), and standard necropsy itself detected only 55% of infected badgers (31). This combination of imperfect capture success, low average infection prevalence, and imperfect test sensitivity means that the numbers of badgers to be killed by selective culling would probably be low, usually just one or two badgers within a social group (32). The same factors, combined with incomplete vaccine efficacy (33), mean that some infected and some susceptible badgers would be expected to remain despite implementation of TVR.Simulations indicate that the likely consequences of TVR for cattle TB control are highly sensitive to assumptions about whether culling small numbers of badgers prompts social perturbation (34, 35). Neither cage trapping for testing nor vaccination has been found to cause behavioral change. If the culling component of TVR likewise causes no perturbation, then TVR is predicted to reduce the prevalence of M. bovis infection in badgers and hence the incidence of cattle TB (34, 35). However, if TVR causes perturbation similar to that associated with past large- and small-scale culling, then it is projected to prompt sustained (34) or transient (35) increases in cattle TB. Unfortunately, it is not known which of these scenarios is more likely. Although the behavioral and epidemiological consequences of nonselective culling are relatively well understood, there have been no empirical studies of badgers’ behavioral responses to killing small numbers of animals per social group, as would occur under TVR and other forms of selective culling.In this paper, we use data from a large-scale study to assess whether killing small numbers of badgers would be expected to prompt social perturbation. We compare patterns of badger movement and M. bovis infection at the start of the RBCT (conducted 1998–2005) (14) with two indices of badger mortality. Our first measure, road density, provides an index of the numbers of badgers killed in road accidents (36), an important cause of badger mortality in Britain (37, 38). Our second measure is prior nonselective culling, conducted during the period 1986–1998 as small-scale badger removal operations (BROs), which typically targeted single farms (8). We hypothesized that high road densities and intense prior culling would each lead to expanded badger movement and elevated M. bovis prevalence. Further, we hypothesized that perturbation might be avoided if the number of badgers killed remained below a certain threshold, and sought to estimate this threshold.     

The impact of alcohol on BCG-induced immunity against Mycobacterium tuberculosis

Background: Alcoholics are at heightened risk for developing active tuberculosis. This study evaluates chronic alcohol consumption in a murine model of vaccination with Mycobacterium bovis Bacille Calmette–Guèrin (BCG) and subsequent pulmonary infection with virulent Mycobacterium tuberculosis. Methods: BALB/c mice were administered the Lieber–DeCarli liquid ethanol diet or pair‐fed the liquid control diet for 3 weeks either before or after subcutaneous vaccination with M. bovis BCG. At least 3 weeks after BCG vaccination, groups of mice on the aforesaid diets were challenged with intratracheal infection with M. tuberculosis H37Rv. Lung mycobacterial burden, and lung and lung‐associated lymph node CD4⁺ lymphocyte production of tuberculosis‐specific interferon (IFN)‐γ were assayed. Popliteal lymph node lymphocytes from both dietary regimens undergoing BCG vaccination (in the absence of M. tuberculosis infection) were also evaluated for purified protein derivative–induced IFN‐γ production by ELISpot assay. Results: Mice begun on alcohol prior to vaccination with M. bovis BCG demonstrated impaired control of pulmonary challenge with virulent M. tuberculosis, as well as impaired lung CD4⁺ and popliteal lymph node T‐cell IFN‐γ responses. If BCG vaccination was delivered prior to initiation of alcohol feeding, the mice remained protected against a subsequent challenge with M. tuberculosis, and BCG‐induced immunity was not impaired in either the lung or the popliteal lymph nodes. Conclusions: Alcohol consumption blunts the development of the adaptive immune response to M. bovis BCG vaccination, which impairs the control of a secondary challenge with M. tuberculosis, but only if the alcohol exposure is begun prior to BCG vaccination. These results provide insight into mechanisms by which alcohol consumption impairs antimycobacterial immunity, including in response to vaccination and subsequent pathogenic challenge.     

BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route

Since studies on cellular immune responses have demonstrated the role of the mucosal lymphoid system of the respiratory tract, we have studied responses obtained from the local respiratory route, compared to the systemic intradermal route, of BCG immunization. Guinea pigs vaccinated with different doses of BCG via both routes served to follow lymphoid cell proliferation, hilar lymph node and lung BCG clearance, lung granuloma formation and protection induced after virulent challenge.Results demonstrate that the aerogenic route of vaccination with BCG has no harmful side-effects for the host. In comparison with the intradermal route of vaccination, aerogenic vaccination with 10⁵ BCG cfu induced higher local cellular immune responses and a substantially improved protective effect.     

Serum bilirubin levels, UGT1A1 polymorphisms and risk for coronary artery disease

Low levels of the antioxidative serum bilirubin are associated with vascular aging and an increased risk for coronary artery disease (CAD). UGT1A1 is the major gene influencing bilirubin concentrations. Therefore, we investigated an association of bilirubin levels and two polymorphisms in the promoter of UGT1A1 (-53(TA-repeat) polymorphism and T-3279G) in 477 patients with premature, familial CAD and 619 age- and sex-matched controls. Bilirubin concentrations were significantly lower in cases than in controls (0.62 ± 0.36 vs. 0.76 ± 0.41 mg/dl for men, p = 1.2 × 10⁻¹⁰; and 0.42 ± 0.29 vs. 0.55 ± 0.23 mg/dl, p = 1.9 × 10⁻⁹ for women). Both polymorphisms showed a strong association with bilirubin levels with higher levels for homozygote carriers of the minor allele. These associations were most pronounced in male controls and patients (p = 5.9 × 10⁻²⁶ and p = 3.4 × 10⁻¹⁶, respectively, for the -53(TA-repeat) polymorphism). Logistic regression analysis revealed low bilirubin levels but not the UGT1A1 polymorphisms to be significantly associated with CAD: OR (95% CI) 0.90 (0.86–0.94), p = 2.6 × 10⁻⁶ for men and 0.77 (0.68–0.87), p = 3.2 × 10⁻⁵ for women, respectively for each 0.1 mg/dl increase of bilirubin. These results indicate that it is rather decreased bilirubin levels in general than the changes in the genetic variation of this gene that increase the risk for CAD.     

Low frequency of moaA3 gene among the clinical isolates of Mycobacterium tuberculosis from Tamil Nadu and Pondicherry – south eastern coastal states of India

Background  

Comparative genomic analysis of M. tuberculosis H37Rv and M. bovis BCG have shown that 16 RDs (Regions of Differences) are deleted in BCG and have shown six deletion regions in M. tuberculosis H37Rv. RD1, is present in M. tuberculosis but is absent in all M. bovis BCG sub-strains. A study from Kerala, a south-western coastal state of India aimed to find out differences in RD1 region showed for the first time the presence of moaA3 gene in majority of their clinical isolates, that was absent in type strain H37Rv. We attempted to find out such polymorphism between type strains and the clinical isolates within RD1, targeting moaA3 gene among the clinical isolates of Tamil Nadu & Pondicherry, south-eastern coastal states of India     

Mitochondrial free calcium regulation during sarcoplasmic reticulum calcium release in rat cardiac myocytes

Cardiac mitochondria can take up Ca²⁺, competing with Ca²⁺ transporters like the sarcoplasmic reticulum (SR) Ca²⁺-ATPase. Rapid mitochondrial [Ca²⁺] transients have been reported to be synchronized with normal cytosolic [Ca²⁺]_i transients. However, most intra-mitochondrial free [Ca²⁺] ([Ca²⁺]_mito) measurements have been uncalibrated, and potentially contaminated by non-mitochondrial signals. Here we measured calibrated [Ca²⁺]_mito in single rat myocytes using the ratiometric Ca²⁺ indicator fura-2 AM and plasmalemmal permeabilization by saponin (to eliminate cytosolic fura-2). The steady-state [Ca²⁺]_mito dependence on [Ca²⁺]_i (with 5 mM EGTA) was sigmoid with [Ca²⁺]_mito < [Ca²⁺]_i for [Ca²⁺]_i below 475 nM. With low [EGTA] (50 μM) and 150 nM [Ca²⁺]_i (± 15 mM Na⁺) cyclical spontaneous SR Ca²⁺ release occurred (5–15/min). Changes in [Ca²⁺]_mito during individual [Ca²⁺]_i transients were small ( 2–10 nM/beat), but integrated gradually to steady-state. Inhibition SR Ca²⁺ handling by thapsigargin, 2 mM tetracaine or 10 mM caffeine all stopped the progressive rise in [Ca²⁺]_mito and spontaneous Ca²⁺ transients (confirming that SR Ca²⁺ releases caused the [Ca²⁺]_mito rise). Confocal imaging of local [Ca²⁺]_mito (using rhod-2) showed that [Ca²⁺]_mito rose rapidly with a delay after SR Ca²⁺ release (with amplitude up to 10 nM), but declined much more slowly than [Ca²⁺]_i (time constant 2.8 ± 0.7 s vs. 0.19 ± 0.06 s). Total Ca²⁺ uptake for larger [Ca²⁺]_mito transients was  0.5 μmol/L cytosol (assuming 100:1 mitochondrial Ca²⁺ buffering), consistent with prior indirect estimates from [Ca²⁺]_i measurements, and corresponds to  1% of the SR Ca²⁺ uptake during a normal Ca²⁺ transient. Thus small phasic [Ca²⁺]_mito transients and gradually integrating [Ca²⁺]_mito signals occur during repeating [Ca²⁺]_i transients.     

Changes of intra-mitochondrial Ca in adult ventricular cardiomyocytes examined using a novel fluorescent Ca indicator targeted to mitochondria

In this study a Ca²⁺ sensitive protein was targeted to the mitochondria of adult rabbit ventricular cardiomyocytes using an adenovirus transfection technique. The probe (Mitycam) was a Ca²⁺-sensitive inverse pericam fused to subunit VIII of human cytochrome c oxidase. Mitycam expression pattern and Ca²⁺ sensitivity was characterized in HeLa cells and isolated adult rabbit cardiomyocytes. Cardiomyocytes expressing Mitycam were voltage-clamped and depolarized at regular intervals to elicit a Ca²⁺ transient. Cytoplasmic (Fura-2) and mitochondrial Ca²⁺ (Mitycam) fluorescence were measured simultaneously under a range of cellular Ca²⁺ loads. After 48 h post-adenoviral transfection, Mitycam expression showed a characteristic localization pattern in HeLa cells and cardiomyocytes. The Ca²⁺ sensitive component of Mitycam fluorescence was 12% of total fluorescence in HeLa cells with a K_d of  220 nM. In cardiomyocytes, basal and beat-to-beat changes in Mitycam fluorescence were detected on initiation of a train of depolarizations. Time to peak of the mitochondrial Ca²⁺ transient was slower, but the rate of decay was faster than the cytoplasmic signal. During spontaneous Ca²⁺ release the relative amplitude and the time course of the mitochondrial and cytoplasmic signals were comparable. Inhibition of mitochondrial respiration decreased the mitochondrial transient amplitude by  65% and increased the time to 50% decay, whilst cytosolic Ca²⁺ transients were unchanged. The mitochondrial Ca²⁺ uniporter (mCU) inhibitor Ru360 prevented both the basal and transient components of the rise in mitochondrial Ca²⁺. The mitochondrial-targeted Ca²⁺ probe indicates sustained and transient phases of mitochondrial Ca²⁺ signal, which are dependent on cytoplasmic Ca²⁺ levels and require a functional mCU.     

Involvement of Src in L-type Ca channel depression induced by macrophage migration inhibitory factor in atrial myocytes

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that controls inflammatory processes, and inflammation is known to play an important role in the pathogenesis of atrial fibrillation (AF). The present study sought to investigate whether MIF expression is responsible for the changes in L-type Ca²⁺ currents (I_Ca,L) seen in AF. Whole-cell voltage-clamp recordings and biochemical assays were used to study the regulation and expression of I_Ca,L in human atrial myocytes and in HL-1 cells. Basal I_Ca,L was reduced in AF compared to sinus rhythm (SR) controls, mRNA and protein levels of the pore-forming α_1C subunit of L-type Ca²⁺ channel (LCC α_1C) were also decreased, while MIF expression levels were increased in AF. Levels of Src and activated Src (p-Src Y⁴¹⁶) were higher in AF than in SR. Treatment of atrial myocytes from a patient with SR with human recombinant MIF (rMIF) (40 nM, 1 h) was found to depress I_Ca,L amplitudes, while mouse rMIF (20 or 40 nM, 24 h) suppressed peak I_Ca,L in HL-1 cells by  69% and  83% in a concentration-dependent manner. Mouse rMIF impaired the time-dependent recovery from inactivation of I_Ca,L and down-regulated LCC α_1C subunit levels. The depression of I_Ca,L and decrease of LCC protein levels induced by rMIF were prevented by the Src inhibitors genistein and PP1. These results implicate MIF in the electrical remodeling that accompanies AF, probably by decreasing I_Ca,L amplitudes through impairment of channel function, down-regulation of LCC α_1C subunit levels, and the activation of c-Src kinases in atrial myocytes.     

Blood flow and mucoid cap protect against penetration of carcinogens into superficially injured gastric mucosa of rats

In this study we tested the influence of blood flow and the mucoid cap on the penetration of carcinogens to the proliferative cells in the injured rat gastric mucosa. Ten minutes after mucosal exposure to 4.5 mol/liter NaCl,N-[³H]methyl-N-nitro-N-nitrosoguanidine was instilled intragastrically. Hypertonic saline caused superficial mucosal damage, formation of a mucoid cap, high gastric mucosal blood flow, and a large flux of fluid into the gastric lumen. The mean percentage of S-phase cells labeled with carcinogen (the cell population at risk forN-methyl-N-nitro-N-nitrosoguanidine-induced carcinogenesis) in the antrum and corpus was 0.2 and 0.2, respectively, in the injury control group, 10.1 and 2.0 after removal of the mucoid cap, 1.5 and 9.8 after celiac artery ligation, and 28.2 and 21.9 after removal of the mucoid cap and celiac artery ligation. These results show that both the mucoid cap and gastric mucosal blood flow protect against penetration of carcinogens into the superficially injured gastric mucosa.This work was supported by grants from the Norwegian Cancer Society and Helga Sembs fond. Dr Sørbye is a Research Fellow of the Norwegian Cancer Society.     

Attenuated expression of SECIS binding protein 2 causes loss of telomeric reserve without affecting telomerase

The family of selenoproteins have a broad range of functions, including protection against oxidative damage. Previous studies have shown that elevated levels of oxidative damage can induce accelerated loss of telomeric DNA during proliferation of mammalian cells. The incorporation of selenocysteine (Sec) into proteins in mammalian cells requires the Sec insertion sequence (SECIS) binding protein 2 (SBP2). Thus in the present study we have assessed the effect of knocking down the expression of SBP2 on telomere length. Following knock-down of SBP2 expression in two different human cell lines, the MSTO mesothelioma cell line (5 Kb average telomere length) and SY5Y neuroblastoma cell line (4.2 Kb average telomere length), we observed a significant reduction (−0.6 to −1.1 Kb; P  0.01) in telomere length as compared to control cells. This reduction in telomere length was independent of affects on telomerase, since both telomerase activity levels and Tert mRNA expression levels were not altered by knock-down of SBP2 expression. Furthermore, telomeres were particularly sensitive to S1 nuclease digestion following SBP2 knock-down, indicating an increased frequency of oxidative damage-induced lesions in the telomeric DNA in these cells. Together, these observations imply that selenoproteins may help protect telomeric reserve in mammalian cells.     

Dogs vaccinated with gentamicin‐attenuated Leishmania infantum or infected with wild‐type parasite can be distinguished by Western blotting

An attenuated line of Leishmania infantum (the H‐line), developed through exposure to gentamicin, has been shown to protect dogs against canine visceral leishmaniasis. A specific diagnostic test to differentiate dogs vaccinated with the attenuated line from dogs infected with L. infantum wild‐type (L. infantum WT) could be a valuable tool in evaluating the effectiveness of canine vaccination. In this study, 28 healthy dogs were allocated into four groups. In Group I and Group II (eight dogs per group), dogs were immunized subcutaneously (s.c.) with L. infantum H‐line, and the dogs of Group II challenged s.c. with L. infantum WT, at 2 months post‐immunization. In Group III, eight animals were challenged s.c. with L. infantum WT, and four dogs of Group IV were injected s.c. with PBS. We found that sera from vaccinated dogs recognize a 21 kDa antigen of promastigotes of L. infantum H‐line but not of L. infantum WT, whereas sera from unvaccinated dogs challenged with L. infantum WT, recognized a 21 kDa antigen of promastigotes of L. infantum WT but not of L. infantum H‐line. Sera from dogs challenged with L. infantum WT with prior vaccination with L. infantum H‐line, recognized a 21 kDa antigen of both L. infantum WT and L. infantum H‐line. These results suggest that the Western blot analysis of antibodies against 21 kDa antigens of L. infantum H‐line and WT may be a useful technique for distinguishing between dogs vaccinated with L. infantum H‐line and dogs naturally infected with L. infantum WT.     

The immune response in two populations of wild badgers naturally infected with bovine tubercle bacilli

This study describes the immune responses of two defined badger populations; one from East Sussex and another from Staffordshire. The mean in vitro lymphoproliferative response, of all infected badgers from both areas, to Glaxo BCG, was significantly greater than that of healthy animals. The infected badgers had significantly higher antibody levels against mycobacterial antigens, especially New Tuberculin, than did the healthy animals. All the healthy and tuberculous badgers from the Staffordshire area were invariably unreactive to the various preparations used for skin-testing. However, in the East Sussex area, positive reactions were obtained in 10 out of 37 healthy and 7 out of 10 infected animals. This is the first account of positive skin tests in free living badgers. These results support the concept that badgers infected with bovine tubercle bacilli pass through an immunological spectrum throughout much of which they are unlikely to be important sources of infection. In the early stages, tubercle bacilli are excreted from infected wounds, whereas in the later stages, failure of cell-mediated immunity results in excretion of tubercle bacilli from other sites and the badger becomes a potent source of infection.     

Reduced DIDS-sensitive chloride conductance in Ae1-/- mouse erythrocytes

The resting membrane potential of the human erythrocyte is largely determined by a constitutive Cl⁻ conductance  100-fold greater than the resting cation conductance. The 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive electroneutral Cl⁻ transport mediated by the human erythroid Cl⁻/HCO₃⁻ exchanger, AE1 (SLC4A1, band 3) is > 10,000-fold greater than can be accounted for by the Cl⁻ conductance of the red cell. The molecular identities of conductive anion pathways across the red cell membrane remain poorly defined. We have examined red cell Cl⁻ conductance in the Ae1^−/− mouse as a genetic test of the hypothesis that Ae1 mediates DIDS-sensitive Cl⁻ conductance in mouse red cells. We report here that wildtype mouse red cell membrane potential resembles that of human red cells in the predominance of its Cl⁻ conductance. We show with four technical approaches that the DIDS-sensitive component of erythroid Cl⁻ conductance is reduced or absent from Ae1^−/− red cells. These results are consistent with the hypothesis that the Ae1 anion exchanger polypeptide can operate infrequently in a conductive mode. However, the fragile red cell membrane of the Ae1^−/− mouse red cell exhibits reduced abundance or loss of multiple polypeptides. Thus, loss of one or more distinct, DIDS-sensitive anion channel polypeptide(s) from the Ae1^−/− red cell membrane cannot be ruled out as an explanation for the reduced DIDS-sensitive anion conductance.     

Role of prostaglandins in the early phases of experimental gastric carcinogenesis in the rat

Summary The study was initiated to evaluate the effect of N-methyl-N-nitro-N-nitrosoguanidine (NG) on gastric intraluminal prostaglandin release during a 30-day treatment period and to investigate the effect of a stable prostaglandin E¹ analogue (misoprostol) on NG-induced gastric mucosal damage during the same time period. Samples of gastric juice (1 h) were obtained from 40 male Sprague-Dawley rats with chronic gastric fistulas, in basal conditions and after 5, 15 and 30 days of continuous oral administration of NG (120 mg/l) or tap water. Aliquots of gastric juice were titrated with 0.1 M NaOH. Other aliquots were extracted with ethyl acetate and subjected to specific radioimmunoassay for prostaglandin E₂. The severity of gastric mucosal lesions was evaluated in 60 rats after 5 days and 30 days of continuous oral administration of NG (120 mg/l) or NG plus misoprostol (200 g/kg^-1/day^-1) or tap water, and a histological study was carried out. Administration of NG induced a significant decrease of gastric intraluminal prostaglandin E₂ concentration at 15 and 30 days. Oral administration of misoprostol, at non-antisecretory doses, protected the rats against NG-induced gastric mucosal damage. Prostaglandins may be involved in the early phases of experimental gastric carcinogenesis.This work was supported by grants from the Ministry of Education (MPI)     

DNA methylation in the digestive tract of F344 rats during chronic exposure toN-methyl-N-nitrosourea

Summary The formation ofO ⁶-methyldeoxyguanosine (O ⁶-MedGuo) was determined by an immuno-slot-blot assay in DNA of various tissues of F344 rats exposed toN-methyl-N-nitrosourea (MNU) in the drinking water at 400 ppm for 2 weeks. Although the pyloric region of the glandular stomach is a target organ under these experimental conditions, the extent of DNA methylation was highest in the forestomach (185 molO ⁶-MedGuo/mol guanine). Fundus (91 mol/mol guanine) and pylorus (105 mol/mol guanine) of the glandular stomach, oesophagus (124 mol/mol guanine) and duodenum (109 mol/mol guanine) showed lower levels ofO ⁶-MedGuo but differed little between each other. Thus, no correlation was observed between target organ specificity and the extent of DNA methylation. This is in contrast to the gastric carcinogen,N-methyl-N-nitro-N-nitrosoguanidine (MNNG), which preferentially alkylates DNA of the pylorus, the main site of induction of gastric carcinomas by this chemical. In contrast to MNU, the non-enzymic decomposition of MNNG is accelerated by thiol compounds (reduced glutathione,l-cysteine), which are present at much higher concentrations in the glandular stomach than in the forestomach and oesophagus. During chronic exposure to MNNG (80 ppm), mucosal cells immunoreactive toO ⁶-MedGuo are limited to the luminal surface [Kobori et al. (1988) Carcinogenesis 9:2271–2274]. Although MNU (400 ppm) produced similar levels ofO ⁶-MedGuo in the pylorus, no cells containing methylpurines were detectable by immunohistochemistry, suggesting a more uniform methylation of mucosal cells by MNU than by MNNG. After a single oral dose of MNU (90 mg/kg) cells containing methylpurines were unequivocally identified using antibodies toO ⁶-MedGuo and the imidazole-ring-opened product of 7-methyldeoxyguanosine. In the gastric fundus, their distribution was similar to those methylated by exposure to MNNG, whereas the pyloric region contained immunoreactive cells also in the deeper mucosal layers. After a 2-week MNU treatment, the rate of cell proliferation, as determined by bromodeoxyuridine immunoreactivity, was only slightly enhanced in the oesophagus and in the fundus, but markedly in the forestomach and the pyloric region of the glandular stomach. It is concluded that the overall extent of DNA methylation, the distribution of alkylated cells within the mucosa and the proliferative response all contribute to the organ-specific carcinogenicity of MNU.Abbreviations MNU N-methyl-N-nitrosourea - MNNG N-methyl-N-nitro-N-nitrosoguanidine - O ⁶-MedGuo O ⁶-methyldeoxyguanosine - 7-MedGuo 7-methyldeoxyguanosine (imidazole ring open) - BrdUrd bromodeoxyuridine     

Costal BCG osteomyelitis presenting as a tumor

Summary We present the case of an 11-month-old male infant who had been BCG vaccinated as a neonate. At the age of nine months, he developed a localized tumor above his right mamilla. The tumor destroyed the fourth rib and infiltrated the surrounding soft tissue. X-ray and CT scan suggested a malignant neoplasm. Histology, however, showed the typical picture of tuberculosis andMycobacterium bovis (BCG strain) was isolated from the lesion. Thus, the tumor was caused by an unusual presentation of a costal BCG osteomyelitis which was associated with negative findings in the bone scan.

---

BCG-Osteomyelitis der Rippen unter dem Bild eines Tumors

Zusammenfassung Wir berichten über einen 11 Monate alten männlichen Säugling, der als Neugeborener BCG-geimpft worden war. Im Alter von neun Monaten entwickelte sich ein umschriebener, unbeweglicher Brustwandtumor über der rechten Mamille. Der Tumor zerstörte die vierte Rippe und infiltrierte die umgebenden Weichteile. Auf Grund des Röntgenbildes und des CT-Befundes wurde ein Malignom vermutet. Histologisch zeigte sich jedoch das typische Bild einer Tuberkulose, undMycobacterium bovis (BCG-Stamm) wurde aus dem Operationsmaterial angezüchtet. Als Ursache des Tumors fand sich somit eine ungewöhnliche Form von Rippen-BCG-Osteomyelitis, die mit einem negativen Befund im Knochen-Szintigramm einherging.