Recombinant (F1+V) vaccine protects cynomolgus macaques against pneumonic plague.

Cynomolgus macaques, immunised at the 80 µg dose level with an rF1+rV vaccine (two doses, three weeks apart), were fully protected against pneumonic plague following inhalational exposure to a clinical isolate of Yersinia pestis (strain CO92) at week 8 of the schedule. At this time, all the immunised animals had developed specific IgG titres to rF1 and rV with geometric mean titres of 96.83±20.93 µg/ml and 78.59±12.07 µg/ml, respectively, for the 40 µg dose group; by comparison, the 80 µg dose group had developed titres of 114.4±22.1 and 90.8±15.8 µg/ml to rF1 and rV, respectively, by week 8. For all the immunised animals, sera drawn at week 8 competed with the neutralising and protective Mab7.3 for binding to rV antigen in a competitive ELISA, indicating that a functional antibody response to rV had been induced. All but one of the group immunised at the lower 40 µg dose-level were protected against infection; the single animal which succumbed had significantly reduced antibody responses to both the rF1 and rV antigens. Although a functional titre to rV antigen was detected for this animal, this was insufficient for protection, indicating that there may have been a deficiency in the functional titre to rF1 and underlining the need for immunity to both vaccine antigens to achieve protective efficacy against plague. This candidate vaccine, which has been evaluated as safe and immunogenic in clinical studies, has now been demonstrated to protect cynomolgus macaques, immunised in the clinical regimen, against pneumonic plague.

Human immune response to a plague vaccine comprising recombinant F1 and V antigens.

The human immune response to a new recombinant plague vaccine, comprising recombinant F1 (rF1) and rV antigens, has been assessed during a phase 1 safety and immunogenicity trial in healthy volunteers. All the subjects produced specific immunoglobulin G (IgG) in serum after the priming dose, which peaked in value after the booster dose (day 21), with the exception of one individual in the lowest dose level group, who responded to rF1 only. Three subjects, found to have an anti-rV titer at screening, were excluded from the overall analysis. Human antibody functionality has been assessed by quantification of antibody competing for binding to rV in vitro and also by the transfer of protective immunity in human serum into the naive mouse. Human and macaque IgG competed for binding to rV in vitro with a mouse monoclonal antibody, previously shown to protect mice against challenge with plague, suggesting that this protective B-cell epitope on rV is conserved between these three species. Total IgG to rV in individuals and the titer of IgG competing for binding to rV correlated significantly at days 21 (r = 0.72; P < 0.001) and 28 (r = 0.82; P < 0.001). Passive transfer of protective immunity into mice also correlated significantly with total IgG titer to rF1 plus rV at days 21 (r(2) = 98.6%; P < 0.001) and 28 (r(2) = 76.8%; P < 0.03). However, no significant vaccination-related change in activation of peripheral blood mononuclear cells was detected at any time. Potential serological immune correlates of protection have been investigated, but no trends specific to vaccination could be detected in cellular markers.

Short tandem repeat profiling provides an international reference standard for human cell lines.

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

Twenty-four hour urinary nitrate excretion in 48 populations from 30 countries: an ECP-INTERSALT collaborative study.

BACKGROUND: There is considerable interest in the possible role of nitrate in gastric carcinogenesis, but little information on nitrate intake around the world. This is the first study to give comprehensive standardised data on nitrate excretion as a marker of intake, using 48 worldwide population samples. METHODS: Urinary nitrate excretion has been shown to be a valid measure of nitrate intake in people under 50. This report presents data on 24-hour urinary nitrate excretion from urine collections obtained in the INTERSALT study, based on random samples of men and women aged 20-49 from each of 48 population samples in 30 countries. RESULTS: There was large variation in urinary nitrate excretion both within and between samples; within-sample (individual) distributions tended to be skewed towards higher values. Median values of the samples ranged from 0.42 mmol/day (Labrador, Canada) to 3.52 (Beijing, People's Republic of China) in men and 0.44 mmol/day (Colombia) to 3.44 (Beijing) in women. Overall, median values were higher in men than women by 11% on average (higher in men in 37 of 48 population samples). Comparison by geographical region of median values for men and women combined showed relatively low values in the samples in North America and Northern Europe (range 0.46-0.88 mmol/day), slightly higher values in Western Europe and Africa (0.68-1.11), and intermediate to high values in Southern Europe, Eastern and Central Europe and India (0.86-2.47). The highest median values were found in the Far Eastern samples (up to 3.48). Median values in the Central and South American samples ranged from 0.48 mmol/day (Colombia) to 1.37 (Xingu Indians of Brazil, and Argentina). CONCLUSIONS: For the first time, these data give standardized information on urinary nitrate excretion from different geographical regions of the world, and provide a basis for the further exploration of the role of nitrate in the aetiology of disease in human populations.

Comparison of selective agars for the isolation and identification of Klebsiella oxytoca and Escherichia coli from environmental drinking water samples.

Various selective media were assessed for their ability to detect and differentiate Klebsiella oxytoca and Escherichia coli in environmental water samples. Only two, Membrane Lauryl Sulphate agar and Deoxycholate Agar, could differentiate the two coliforms from each other and from the 'background' heterotrophs in water and this was a consequence of E. coli's ability to grow at 44 degrees C and 37 degrees C whereas Kl. oxytoca could only grow at 37 degrees C. Modified M-FC medium effectively differentiated Kl. oxytoca but not E. coli in environmental samples. Other media characterized the different coliforms in pure culture but failed to do likewise in environmental samples. For example, pure cultures of E. coli fluoresced when MUG was added to the medium but single colonies on a mixed species plate failed to do so. MT7 agar distinguished the two coliforms from water heterotrophs but not from each other.

Vertigo: assessment and management.

Elucidation of the cause of vertigo must consist of an adequate history to see if a clinical entity such as BPPV or Meniere's Disease can be recognised, and a general physical examination with emphasis on: The ear--discharge, perforation, fistula sign and hearing. Vestibular system--nystagmus and Romberg test with past pointing and positional testing. CNS--cranial nerves, cerebellar signs and gait. CVS--BP and bruits in neck and cardiac irregularities. Audiogram, ENG and CT or MRI. The specific treatment must be supplemented by sympathetic, symptomatic therapy and reassurance that the condition is not a mystery but clearly defined. The probable cause and lines of treatment available can be explained.

Significant endogenous synthesis of nitrate does not appear to be a feature of influenza A virus infection.

There is much concern about the role of nitrate in the formation of carcinogenic N-nitroso compounds. There has been renewed interest in the endogenous formation of nitrate arising as a host response to infection. This study was designed to investigate whether the large increases in nitrate excretion rate reported (6-15-fold) for certain infectious diseases is also a feature of systemic influenza infections. Volunteers were challenged either with an attenuated strain of influenza A virus or with saline; and excreted nitrate was measured in subsequent 24-h urine samples. Both with and without adjustment for potential confounding by dietary and other factors, it was clear that neither mild nor moderate influenza A virus infection resulted in substantial endogenous nitrate biosynthesis since all the variation in urinary nitrate excretion observed was within the range of normal daily fluctuations. It remains possible that a stronger and more consistent nitrate excretion response might be observed in other infectious illnesses with greater systemic disturbance.

Nitrate and N-nitrosoproline excretion in two Italian regions with contrasting rates of gastric cancer: the role of nitrate and other factors in endogenous nitrosation.

Exposure to nitrate and propensity for endogenous nitrosation were examined in 80 healthy males, aged 25-40 years, residing in areas of Italy with long-standing high (Florence) and low (Cagliari) rates of gastric cancer. Nitrate exposure was assessed by measurement of urinary nitrate excretion over 12 hr, and endogenous nitrosation was assessed using the N-nitrosoproline test (NPRO-test). Our hypothesis was whether the geographic variation in cancer rate correlated with nitrate exposure or nitrosating ability. Exposure to background sources of NPRO was significantly higher in the high-risk subjects (phi = 0.04) whereas no differences were found in exposure to nitrate or in urinary NPRO levels after L-proline loading (test NPRO levels). The regional difference in test NPRO was almost completely accounted for by background NPRO exposure. Examination of individual rather than grouped data revealed that exposure to nitrate was a major factor in NPRO formation. No other factors studied (age, dietary-questionnaire-assessed intake of anti-oxidant vitamins) had a significant effect. Geographical variation in gastric cancer risk did not, therefore, correlate with either nitrate exposure or propensity for endogenous nitrosation of L-proline.

The N-nitrosoproline test as a measure of cancer risk in geographical comparison studies: results from Italy and an overall comparison.

The N-nitrosoproline (NPRO) test has been used in studies in which populations at high risk of cancer have been compared with equivalent populations at lower risk, to examine whether the geographical variation in cancer risk correlates with propensity for endogenous nitrosation, as assessed by the NPRO test. The usual method employed has been to determine NPRO in 12- or 24-h urine samples, after ingestion of L-proline, in a representative sample of the general population. We present results from one such geographical study conducted in two regions of Italy (Florence and Cagliari) with an approximately three-fold variation in gastric cancer mortality. The nonsignificant difference in mean NPRO excretion between the two populations was insufficient to explain the difference in cancer risk. The fact that there are appreciable international differences in formation of NPRO suggests, firstly, that nitrosation may be of relevance to cancer risk in some countries but not in others and, secondly, that variations within one country may not be large enough for significant geographical differences to be evident. Multivariate analysis of individual, rather than grouped, results from our Italian study made it possible to quantify the relevance of different factors to NPRO formation: a major factor is exposure to nitrate. Important relationships may be missed by analysing only grouped data.

N-nitrosoproline excretion in the presence and absence of gastric disease.

N-nitrosoproline (NPRO) excretion, an indicator of endogenous nitrosation, was measured in a group of hospital inpatients who were identified by endoscopy and gastric biopsy as either having gastric lesions or having healthy stomachs. NPRO was assayed in background 24-hour urine samples and samples collected after loading doses of nitrate and L-proline. The presence of gastric lesions was associated with altered gastric pH and concomitant changes in gastric juice nitrate and nitrite concentration. Gastric juice pH increased with increasing severity of gastric disease (P = 0.031) and patients with normal stomachs had a lower gastric pH than those with chronic atrophic gastritis (CAG) (3.0 vs. 6.5, P = 0.017). The changes in gastric juice nitrate concentration were in the reverse direction (P = 0.002 for trend) with normal patients having higher mean levels than CAG patients (12.7 vs. 5.5 micrograms/ml, P less than 0.0001). Nitrite concentration increased with severity of gastric disease but the results were not significant (normal, 82.9 vs. CAG, 223.4 ng/ml, P = 0.069). No association was found between the presence of gastric lesions and increased urinary NPRO excretion. Mutagenic activity was not detected in any of the gastric juice samples.

The effect of gastric achlorhydria on the urinary recovery of nitrate in man: relevance to urinary nitrate as a measure of dietary nitrate exposure.

It is important to have a reliable method of assessing the dietary nitrate exposure of populations for a proper understanding of the potential health effects of the endogenous metabolites of this ion to be gained from epidemiological studies. Recently we strongly advocated the use of the nitrate analysis of 24 h urine samples as being a superior method for such studies. Our previous observations and those of others relating to nitrate pharmacology in healthy human volunteers formed the basis of this judgement. The purpose of this study was to determine whether gastric achlorhydria or hypochlorhydria has any significant gross effects on the urinary recovery of dietary nitrate and to what extent the inclusion of such individuals would compromise the results of potential epidemiological studies. The results demonstrated a significantly greater loss of dietary nitrate as measured by urinary recovery in achlorhydrics than in normochlorhydrics, presumably as a consequence of bacterial metabolism in the colonized stomach. Thus the average urinary nitrate recovery of a 1.5 mmol challenge was 33% in individuals with reduced stomach acidity as compared to 56% in normal controls. This significant further loss of nitrate (average 23%) when intragastric conditions favour bacterial colonization clearly indicates that for valid assessments and comparisons of nitrate exposure between populations it would be wise to exclude individuals with low levels of stomach acid where this is likely to lead to significant gastric colonization by nitrate-reducing bacteria (i.e. pH less than 4-5).

The effect of different sources of nitrate exposure on urinary nitrate recovery in humans and its relevance to the methods of estimating nitrate exposure in epidemiological studies.

It was demonstrated that nitrate concentration fluctuates dramatically in both urine and saliva throughout the study period and hence single time point ('spot') samples of either fluid give a poor measure of previous nitrate exposure. In contrast, the nitrate recovery in complete 24 h urine collections showed a strong positive correlation with previous nitrate exposure which was independent of the nature of the food matrix in which the nitrate was ingested. Regression analysis of the data showed that the apparent urinary recovery of 70% appears to consist of 55% arising from the original challenge and a background of approximately 0.22 mmol/day which is independent of the challenge and which may reflect endogenous mammalian synthesis of nitrate. It is therefore important that in future epidemiological studies, in which dietary nitrate exposures are to be determined, that analyses are conducted only on 24 h urine collections. A large inter-individual variation was found in the conversion of salivary nitrate to nitrite presumably mediated by the oral flora. The resulting differences in nitrite formation may be one of the factors determining the level of endogenous N-nitroso compound formation in an individual.

N-nitrosoproline excretion in patients with gastric lesions and in a control population.

N-Nitrosoproline (NPRO) excretion was measured in a group of hospital in-patients who were identified as either bearing gastric lesions or having apparently healthy stomachs. NPRO was assayed in background 24-h urine samples and then in urines collected after loading doses of nitrate and proline. The presence of gastric lesions was associated with altered gastric juice pH and nitrite concentration, but not with NPRO excretion. The significance of NPRO excretion as a marker of endogenous nitrosation is dependent on the interpretation of this result.