Kernel-based learning algorithms are among the most advanced machine learning methods and have been successfully applied to a variety of sequence classification tasks within the field of bioinformatics. Conventional kernels utilized so far do not provide an easy interpretation of the learnt representations in terms of positional and compositional variability of the underlying biological signals.degree of position-dependency, our feature space representation can be tailored to the characteristics of the biological problem at hand. A regularized learning scheme enables application even to biological problems for which only small sets of example sequences are available. Our approach includes a visualization method for transparent representation of characteristic sequence features. Thereby importance of features can be measured in terms of discriminative strength with respect to classification of the underlying sequences. To demonstrate and validate our concept on a biochemically well-defined case, we analyze E. coli translation initiation sites in order to show that we can find biologically relevant signals. For that case, our results clearly show that the Shine-Dalgarno sequence is the most important signal upstream a start codon. The variability in position and composition we found for that signal is in accordance with previous biological knowledge. We also find evidence for signals downstream of the start codon, previously introduced as transcriptional enhancers. These signals are mainly characterized by occurrences of adenine in a region of about 4 nucleotides next to the start codon.We propose a kernel-based approach to datamining on biological sequences. With our method it is possible to model and analyze positional variability of oligomers of any length in a natural way. On one hand this is achieved by mapping the sequences to an intuitive but high-dimensional feature space, well-suited for interpretation of the learnt models. On the other hand, by means of the kernel trick we can provide a general learning algorithm for that high-dimensional representation because all required statistics can be computed without performing an explicit feature space mapping of the sequences. By introducing a kernel parameter that controls the We showed that the oligo kernel can provide a valuable tool for the analysis of relevant signals in biological sequences. In the case of translation initiation sites we could clearly deduce the most discriminative motifs and their positional variation from example sequences. Attractive features of our approach are its flexibility with respect to oligomer length and position conservation. By means of these two parameters oligo kernels can easily be adapted to different biological problems. Different concepts of statistical pattern analysis and modelling are successfully used for this purpose. Sophisticated methods like Hidden Markov Models , neural et al.  proposedoligo kernels which provide a novel approach to datamining on biological sequences, based on the powerful concept of kernel feature spaces [a) the existing methods provide either position-dependent representations based on mononucleotide occurrences [K-mer occurrences restricted to a completely position-independent representation [K-mer representations may also be realized by the oligo kernel. (b) Furthermore, our method provides an intuitive visualization approach to present relevant oligomers and their positional variability to the user. For that purpose a suitable measure of discriminative power can be utilized to score different motifs according to their relevance for classification. Oligo kernels can therefore be applied to infer characteristic sequence features which, in turn, can be used to identify functionally important signals. This property qualifies the oligo kernel as a useful datamining tool for the analysis of biological sequences.Herein, we introduce e spaces . Our appurrences ,7 or theentation ,9. By coEscherichia coli K-12 because a reliable set of biochemically verified sites is available for that case. Prediction of TIS is not satisfactorily solved for prokaryotic genes [end position of a predicted gene, it is by no means trivial to determine the corresponding start position as start codons are not unique; they are also used to code for amino acids inside genes. Systematic studies have shown that existing gene-prediction programs perform poorly when it comes to predicting the correct TIS [To demonstrate the usefulness of our approach, we applied oligo kernels to analyze translational initiation sites (TIS) for ic genes . For bacrect TIS ,12. ConsAlthough our method is based on classification of sequences we do not intend to provide a TIS prediction tool in this paper. On one hand the method provides a general tool for identification and characterization of signals in biological sequences which is not restricted to TIS sequences. On the other hand our datamining approach may be viewed as a prior step for constructing TIS predictors. By means of an easy visual interpretation of the inferred model it can provide new insights which in turn can steer the construction of efficient predictors. We expect that this feature will in particular be useful for analysis of prokaryotic genomes for which only small sets of experimentally verified TIS exist. a kernel is simply a function . It implicitly applies a usually nonlinear transformation  to elements x, x' in input space  and then computes the inner ("dot") product within a resulting feature space :Kernel-based learning  providesk(x, x') = Φ(x)·Φ(x').     (1)x and x' which is defined as an inner product of the feature space objects Φ(x) and Φ(x'). The concept of kernel-induced feature spaces may first be motivated by the objective to provide an adequate representation for the data, which is more suitable for the classification task at hand than the original input space . Second, the so-called kernel trick suggests to perform learning in that feature space without any explicit computation of the mapping Φ, just using inner products of the feature space representatives. The inner products in turn may be computed by some realization of the above kernel function (1) in a hopefully efficient way. That trick may prove useful in situations where the feature space objects are rather high-dimensional and computation of Φ is costly. As will be shown below, these objects may even be functions, i.e. objects of infinite dimensionality.A kernel can be viewed as a similarity measure for the input space objects w ∈  and an scalar offset b. With an explicit feature space mapping of the input elements the primal form of the discriminant isFor construction of a linear classifier in feature space the discriminant for the two-class problem requires a feature space weight vector f(x) = w·Φ(x) + b.     (2)x to one of the two classes, i.e. to realize a binary classifier. By means of the kernel trick the above primal form can be replaced by its dual representation, which does not require access to any feature space elements. The basis for constructing a kernel classifier is a training set of n labelled input space examples x1, x2,...,xn. Using these examples, we can construct a linear discriminant in feature space without explicit computation of the feature space representatives. Instead we only compute inner products according to the above kernel function weighted by parameters αi in order to get the dual form of the discriminant:The sign of that discriminant can be used to assign b = 0 for convenience. The weights αi determine how much individual training examples contribute to the discriminant. Optimal values have to be determined by some suitable learning algorithm which will be outlined below.where we assumed oligo functions and oligo kernels we achieve both: learnability and interpretability of the proposed model. For a suitable realization of the primal form, the idea is to represent oligomer (k-mer) occurrences in terms of smooth functions which preserve positional information about oligomer locations to an adjustable extent. In order to model positional variability of the underlying biological signals we introduce some measure of positional uncertainty. The degree of positional uncertainty is controlled by some smoothing parameter σ which allows the model to be selected from a continuous space of candidates between the two extreme cases of completely position-dependent and position invariant models, respectively. An important advantage of the oligo kernel over previous position-dependent kernels is that, besides the dual representation of the feature space discriminant which is suitable for learning, it also provides a primal representation which is suitable for interpretation of the learnt discriminant. Therefore, classifiers based on oligo kernels not only provide a method for predicting signals in sequences but they may also help to identify relevant K-mers of any length and to analyze their positional variability. The two kinds of representations are detailed in the following.Unlike most SVM approaches which just propose a kernel and do not care about the primal form of the discriminant, i.e. about interpretability, we here first propose a suitable primal representation and then apply the kernel trick in order to provide a general learning scheme for that discriminant. By means of the primal/dual-concept of oligo functions which encode occurrences of oligomers in sequences with an adjustable degree of positional uncertainty. For that purpose, occurrences are not represented by exact ("hard") assignment of the oligomers to their observed positions, but rather by some kind of "fuzzy" assignment according to the assumed positional uncertainty. Therefore oligo functions can be viewed as fuzzy membership functions [K-mer occurrences to positions in a "soft" manner. For a convenient realization we choose these functions to be mixtures of Gaussians with the variance σ2 of the Gaussians controlling the degree of positional uncertainty. Thus, for an alphabet  and a sequence s which contains K-mer ω ∈  at positions Sω = {p1, p2,...} we obtain the oligo functionFor the primal representation of the feature space discriminant (2) we introduce the concept of unctions , up to at which does not need to be restricted to a discrete domain so far. The smoothing parameter σ adjusts the width of the Gaussians which are centered on the observed oligomer positions and therefore it determines the degree of position-dependency of the function-based feature space representation. While small values for σ imply peaky functions, large values imply flat functions with the limiting case σ → ∞ preserving no positional information at all. The effect of positional uncertainty is shown in figure s the occurrences of all K-mers contained in  = {ω1, ω2,...,ωm} can be represented by a vector of m oligo functions which yields the final feature space representation of that sequence:with continuous position variable Note, that the feature space objects are vector-valued functions, which can be stressed using the following notation:φs(t) = [μ1(t), μ2(t),..., μm(t)]T     (6). Thus, the feature space representatives are curves in the m-dimensional space of all K-mers.where we used the shortcut φs(ti) of function values for all positions ti considered. Then, usual vector-based learning algorithms could be applied in order to infer relevant sequence characteristics. Unfortunately, this approach would only be feasible for short oligomers, i.e. for small K, because already for DNA sequences with l positions the number of required vector dimensions is l × 4k while for protein sequences it is l × 20K. In our application we used l = 200 positions and therefore we would have got nearly a million dimensions for representation of hexamer occurrences, which is prohibitive for usual vector-based learning schemes.If we discretize the oligo functions according to the actual number of sequence positions, we would be able to assemble feature vectors of finite dimensionality, just stacking the vectors αi we can construct the vector-valued weight function of the discriminant:While oligo functions in general cannot be used directly for learning of a feature space discriminant they are well-suited for the interpretation of a learnt discriminant. The latter fact is an important prerequisite for our datamining approach which provides an intuitive insight of the kernel-based sequence model. Considering the primal form of the feature space discriminant, the weight vector becomes a vector-valued function arising from a linear combination of the feature space representations of the sequences. With the learnt parameters m-dimensional space of oligomers. For each of the m components we have a linear combination of oligo functions where the weights αi determine the contribution from each of the n training sequences. Due to its primal representation based on oligo functions, an oligo kernel classifier provides an intuitive insight into its discriminant because high positive (negative) values of the weight function contribute to prediction of the positive (negative) class. So the user can easily inspect the learnt representation which in turn allows him to draw conclusions about the relevance of certain oligomers and their locations in terms of their discriminative power.which is a curve in the w(t) = [w1(t), w2(t),..., wm(t)]T according to their L2-normIn order to facilitate the interpretation of the discriminant, one may restrict the analysis to the most discriminative oligomers. This can be done by ranking the component weight functions of Because higher norms indicate a more important role in discrimination, selection of the corresponding weight functions helps to keep the focus on the relevant oligomers. A similar criterion has also been suggested for the position-independent spectrum kernel in order to identify discriminative motifs which can be used to distinguish real exons from pseudo exons .visualization of these discriminative weight functions provides complete access to the information which has been extracted from the data. For an overview, all discriminative weight functions wi may be discretized and stored in a matrix which may be visualized as a bitmap image using grey values or color to encode the function values. Using l discrete sequence positions ti, the m × l image matrix can easily be obtained asBecause the feature space weight vector can be represented as a vector of functions, a suitable W = [w(t1),w(t2),...,w(tl)].     (9)L2-norm. For a closer look on the role of single oligomers the corresponding weight functions can be visualized as usual 2D function plots. Examples for both kinds of visualization will be shown in the section on results.For longer oligomers only a subset of weight functions may be used, which can be selected according to the above si from our dataset is represented by a vector of oligo functions, i.e. a curve  in the m-dimensional space of all K-mers. While this feature space representation is well-suited for interpretation of the discriminant, it is impractical for learning the discriminant from sequence data. For that purpose we shall utilize the dual form of the discriminant, based on oligo kernels. By means of the kernel trick the dual representation is well-suited for training and any kernel-based learning algorithm [ an inner product of two sequence representations φi, φj can be defined asIn the previous section we have derived a feature space representation for our biological sequences, where a certain sequence lgorithm  may be ud = |p - q| between two oligo positions p and q. This function I(d) is equivalent to the convolution of two Gaussians with equal variance. Up to some constant factor, the convolution of two Gaussians is a Gaussian whose variance is the sum of the original variances. Therefore the integral can be calculated according toNote that the second integral is a function of the distance Replacing the second integral in (10), the oligo kernel, i.e. the inner product of two feature space representatives, can be computed according toσ. For the limiting case σ → 0 with no positional uncertainty, only oligomers which occur at the same positions in both sequences contribute to the sum. In general it would not be appropriate to represent oligomer occurrences without positional uncertainty, which would imply zero similarity between two sequences if no K-mer appears at exactly the same position in both sequences. Regarding the other extreme σ → ∞ with maximum positional uncertainty, position-dependency of the kernel completely vanishes: all terms of oligomers, occurring in both sequences, contribute equally to the sum, regardless of their distance. Together with the normalization which is introduced below, in the latter position-independent case the oligo kernel becomes identical to the so-called spectrum kernel [From the above definition of the oligo kernel, it is easy to see the effect of the smoothing parameter m kernel  which hal1 and l2, respectively, the above oligo kernel (12) can be computed by evaluation of at most l1 × l2 exponential functions. For a speed-up of the computations these evaluations may be realized by fast table lookups. Fortunately, the maximum number of l1 × l2 evaluations is hardly reached in practice, because we only have to compute terms of (12) for oligomers occurring in both sequences. Only in cases where oligomers often occur at many positions in both sequences the complexity is rising towards O(l1 × l2). These cases become more unlikely for longer oligomers and therefore, with an efficient implementation, the computational cost rapidly decreases with increasing oligomer length K.Regarding computational complexity of the oligo kernel, for two sequences of length L2-norm. Therefore we compute the normalized oligo kernel according toAccording to the above derivation, the feature space representations may have different norms. Here, for the representation of biological sequences, the norm of a feature space object roughly corresponds to the absolute count of the oligomer occurrences. In order to improve comparability between sequences of different length, the feature vectors should be normalized to unit n labelled example sequences  = {(s1,y1), (s2,y2),...,(sn,yn)} with yi = 1 or yi = -1 for positive and negative examples, respectively. Because we want to propose a general learning scheme which is even applicable to problems with small data sets, a regularized learning scheme is essential in order to prevent overfitting. Many regularized learning schemes are available, among which soft-margin SVMs [n linear equations, as shall be outlined below. Given the set  containing n labelled sequences, with labels yi ∈ {-1,1} contained as components in n-vector y, we shall now train a kernel classifier based on discriminant (3) using the proposed oligo kernel. With the kernel matrix  which contains all possible inner products of the training set examples in feature space, we realize a classifier by minimizing the λ-penalized prediction errorThe basis for learning a kernel classifier is a training set of gin SVMs  are usuagin SVMs , which hα = [α1,...,αn]T. By means of the regularization parameter λ > 0 the penalty controls the norm of the feature space discriminant in order to avoid overfitting. Bounding the norm of the discriminant restricts the learning algorithm to put higher weights only on "effective" features which are important for classification. In that way learning is forced to focus on that task-specific information which can actually be drawn from the data. Choosing the parameter vector α to yield a minimum of the above error, an optimal realization can be found by solving the following system of linear equations:with respect to parameter vector K + λnI)α = y     (15) the computational cost of the learning algorithm is rapidly increasing for an increasing training set. In practice we observed that effective routines for matrix inversion enable training with a few thousands of examples. However, on one hand this behavior is a general shortcoming of kernel methods and recently several approximations have been suggested, which can effectively decrease the computational cost [where nal cost ,18. WithE. coli genes from the EcoGene database [E. coli genome of those tools integrated into YACOP [In order to create a reliable dataset we utilized database  and consto YACOP : We founK-mers of length K = 1,...,6. For a reliable estimate of the prediction error we performed 50 runs for each oligomer length where each run comprises training, validation for adjusting the hyperparameters σ and λ and final testing on data which have not been used to adjust the parameters of the classifier. The latter was done on a test set which contained one third of the data. From the remaining data two thirds were used for training and one third for validation of the classifiers with respect to hyperparameter values. Thus, for each run the data were randomly partitioned into training, validation and test sets of size 631, 378 and 567, respectively. For validation we varied the smoothing parameter σ ∈ {0.5, 0.75, 1, 1.5, 2} and the regularization parameter λ ∈ {0.1·0.9i|i = 0, 1,..., 100} in order to minimize classification error on the validation set. With the optimal hyperparameter values we then trained a classifier on the union of training and validation set. Finally that classifier based on optimal hyperparameters was evaluated on the test set to yield the final classification error. The mean test error over the 50 runs together with the corresponding standard deviation is shown in table σ over the 50 runs. The table shows that the lowest error rate of 8.9 percent has been achieved for the 3-mer kernel with a mean value 1.25 of the hyperparameter σ which models the positional variability of the oligomers. This result indicates that the best representation does not necessarily require the lowest positional variability. Note that the mean error is monotonically increasing for decreasing oligomer lengths below and for increasing lengths above the best length 3. Therefore we did not try to model the occurrences of K-mers for K > 6. To investigate the effect of an enlarged data set, we chose additional TIS sequences according to GenBank annotations to yield a data set with about four times the number of examples of our original EcoGene-based set. We included all non-hypothetical coding sequences from the E. coli (U00096) dataset, including those from the EcoGene dataset, as positive examples. The negative examples were generated in the same way for the EcoGene-based set. So we obtained an enlarged set of 2980 positive and 3968 negative examples. The enlarged set was randomly partitioned, as described above, with the same proportions of training, validation and test sets. The average performance over 20 runs with different partitions is shown in table To analyze the TIS sequences by means of the proposed oligo kernel, first of all we tested the predictive power of the feature space representation as it depends on different oligomer lengths. In order to compare their discrimination performance, we trained classifiers using oligo kernels according to W in equation (9) which contains the discretized functions of the primal form weight curve (7) as rows. The image contains the discriminative functions as horizontal lines, with one line for each oligomer. The color of a pixel indicates the level of the corresponding function at each integer position within a window of length 200 nt. Figure W-matrices from the above 50 runs using the trimer kernel. The complete matrix of function values is scaled to yield a unit maximum which is attained by the ATG-function at position 0. In addition, for noise reduction all matrix elements with an absolute value below 0.1 are set to zero. In figure K = 3 can be found on the web page [For interpretation of the learnt TIS models, we applied the visualization techniques which have been described above. To visualize the feature space discriminant we first generated an overview bitmap image of the matrix K-mers for the kernel-based TIS prediction, we computed the L2-norm in equation (8) for all oligomer-specific weight functions of the learnt discriminants. The ten most discriminative K-mers are identified using the average norm over the above 50 runs. The resulting rankings for K = 3,...,6 are depicted as bargraphs in figure K-mer weight function and has been scaled to yield a unit maximum height for each K. For the monomer kernel we found that A is most discriminative followed by G, T, C in decreasing order of the norm. For dimer occurrences the most discriminative oligomer is GG followed by GA, AG, AT, TT and AA, again in decreasing order. For the longer K-mers depicted in figure In the following we use the terms monomer, dimer etc. instead of the more specific terms mononucletide dinucleotide etc. In order to identify the most important From these motifs and the associated weight functions we can conclude that C or T at position -1 and A at position 3 seem to be characteristic for TIS as they show high positive peaks at the corresponding positions . From the pentamer and hexamer occurrences one can deduce a preference for AAA or GCT as a second codon. Start codons GTG and TTG are not characteristic for TIS and therefore the discriminative weight functions of these trimers show high negative peaks at position 0 which can be seen from the overview matrix image of figure On the other hand oligomers contained in AAGGAGA or GAGGAGA have high rank. The corresponding discriminative functions usually put high positive weights on regions located about 10 nt upstream the start codon , and with a combined oligo kernel (OK1...6) which incorporates all K-mers for K = 1,...,6, simply by adding the six different kernels. With respect to the primal representation of the discriminant, adding the kernels means to stack the vectors of oligomer-specific weight functions for different K-mer lengths. Obviously this implies an augmented feature space which might combine the advantages of representations based on short and long oligomers. For each of the six added kernels we chose the smoothing parameter according to the median of the optimal values obtained from the 50 previous runs with single oligo kernels which resulted in values [0.5, 0.5, 1.0, 1.0, 1.0, 1.0] for the length-specific smoothing parameters [σ1,...,σ6].In order to compare our approach with standard methods for modelling of sequence sites, we utilized inhomogeneous, i.e. position-dependent, Markov models which are widely used for the estimation of positional weight matrices. The probabilistic models can easily be estimated from the data and do not require any hyperparameter optimization. For evaluation we utilized one model for each of the two classes (positive/negative) and assigned a sequence to that class with highest probability of the corresponding model. In the case of zero probabilities for both models, as a tie-breaking rule we assigned the sequence to that model with the smallest number of zero probability positions. Classification was considered correct if the model with the smaller number was associated with the right class. For training we estimated the position-specific oligomer probabilities of the model using two thirds of the data while testing was performed on the remaining third. Prediction performance on the test set was again averaged over 50 runs with different random partitions of the data. For comparison with a position-independent kernel we utilized the spectrum kernel (SK) which can be obtained as a special case of the oligo kernel for K-mer length 2 failed to discriminate TIS correctly for nearly half of the data. With an average error rate of 44.6%, performance was not much better than classification by chance, indicating the importance of position information for TIS prediction. With the combined kernel OK1...6 our method could successfully exploit the combination of different K-mer representations and slightly improved the performance of the trimer kernel. In addition, table 1) and dimer (MM2) occurrences, respectively, performed equally well. We found that for the data at hand the 0th order Markov model could not be improved using higher order Markov models. Using Markov models above order 1 the performance even broke down, with a resulting error of ≈ 30%. Therefore in our case the Markov model based representations do not provide an adequate tool for analyzing occurrences of oligomers above length 2. As shown in table K = 1. While theoretically the oligo kernel should not be worse, in practice it is difficult to find the optimal hyperparameters for best discrimination. Performing a two-dimensional grid search for smoothing parameter σ and regularization parameter λ, it cannot be expected to find the exact global optimum. On the other hand, for monomer occurrences there seems to be less positional uncertainty, so that the smoothing parameter is not likely to improve the representation anyway. Therefore the oligo kernel does not seem to be the best tool for pure analysis of monomer occurrences.As can be seen from table We have introduced a novel concept of datamining on biological sequences and exemplified its application on the analysis of prokaryotic translational initiation sites.The interpretation of our results makes clear that the most pronounced signal indicating a TIS besides the start codon is the Shine-Dalgarno region . With ouThe analysis of individual oligo functions  we could clearly deduce the most discriminative motifs and their positional variation from example sequences. These findings directly influence the design of tools for TIS prediction e.g. with respect to the length of up- and downstream regions that have to be analyzed. Attractive features of our approach are its flexibility with respect to oligomer length and position conservation. By means of these two parameters oligo kernels can easily be adapted to different biological problems. We showed that the position independent spectrum kernel can be viewed as a special case of the oligo kernel and that for the analysis of TIS sequences the incorporation of position information is crucial. In contrast to other position-dependent sequence kernels our approach not only provides learnability of a suitable model but also an easy interpretation of the learnt representation.PM designed, implemented and tested the oligo kernel and the associated visualization method and he drafted most of the manuscript. RM accounted for biological expertise and substantial parts of the draft. MT contributed in biological expertise and prepared the datasets. BM assisted in coordination and manuscript writing. All authors read and approved the final manuscript.


Vasectomy techniques have been the subject of relatively few rigorous studies. The objective of this analysis was to compare the effectiveness of two techniques for vas occlusion: intraluminal cautery versus ligation and excision with fascial interposition. More specifically, we aimed to compare early failure rates, sperm concentrations, and time to success between the two techniques.We compared semen analysis data from men following vasectomy using two occlusion techniques. Data on intraluminal cautery came from a prospective observational study conducted at four sites. Data on ligation and excision with fascial interposition came from a multicenter randomized controlled trial that evaluated the efficacy of ligation and excision with versus without fascial interposition. The surgical techniques used in the fascial interposition study were standardized. The surgeons in the cautery study used their customary techniques, which varied among sites in terms of type of cautery, use of fascial interposition, excision of a short segment of the vas, and use of an open-ended technique. Men in both studies had semen analyses two weeks after vasectomy and then approximately every four weeks. The two outcome measures for the analyses presented here are (a) time to success, defined as severe oligozoospermia, or <100,000 sperm/mL in two consecutive semen analyses; and (b) early vasectomy failure, defined as >10 million sperm/mL at week 12 or later.Vasectomy with cautery was associated with a significantly more rapid progression to severe oligozoospermia and with significantly fewer early failures (1% versus 5%).The use of cautery improves vasectomy outcomes. Limitations of this comparison include (a) the variety of surgical techniques in the cautery study and differences in methods of fascial interposition between the two studies, (b) the uncertain correlation between sperm concentrations after vasectomy and the risk of pregnancy, and (c) the use of historical controls and different study sites. Vasectomy techniques have been the subject of relatively few rigorous studies. The Royal College of Obstetricians and Gynecologists  noted thWe have shown in a randomized controlled trial that fascial interposition can reduce failure rates by about half when the occlusion method is suture ligation with vas excision . HoweverAt an experts' meeting organized by Family Health International (FHI) and EngenderHealth in April 2001, experts reviewed data on vas occlusion techniques, including the preliminary pooled results from the randomized controlled trial of fascial interposition mentioned above. Given the apparently high rate of vasectomy failures in that study, the experts recommended that an observational study be done to assess sequential sperm concentrations after vasectomies with cautery to see if there was a qualitative difference between sperm concentrations after vas occlusion by cautery and sperm concentrations after occlusion by ligation and excision. Some participants suggested that if such a study showed a clear difference in the rate of success or the frequency of apparent recanalizations between cautery and ligation and excision with fascial interposition, then this might provide sufficient evidence for service providers to consider switching to the use of a cautery technique .Based on that recommendation, we conducted an observational study of vasectomy by cautery  and thenThe methods of the fascial interposition and cautery studies have been previously described ,6. In brThe cautery study involved four sites in four countries. It was designed to estimate the effectiveness of cautery as currently performed and to describe the trends in sperm counts after vas occlusion by cautery. The surgeons used their customary cautery occlusion techniques, which differed among the sites. At two sites, surgeons used electro-cautery without fascial interposition: one with and one without excision of a short segment of the vas. At the other two sites, they used thermal cautery with fascial interposition: one with and one without an open-ended technique and excision of a short segment of the vas. Three sites used the no-scalpel approach to the vas. Graphic depictions of the four methods used have been published . Of 400 Both studies included frequent semen analyses, beginning at two weeks after vasectomy. In the fascial interposition study, subsequent semen analyses were conducted every four weeks until a man had provided two consecutive azoospermic specimens, was declared a vasectomy failure, or reached the end of study follow-up at 34 weeks. In the cautery study, after the first sample at two weeks, subsequent semen analyses were conducted at weeks 5, 8, 12, 16, 20, and 24, regardless of semen analysis findings. In both studies, participants were asked to record all ejaculations between semen analyses on a wallet-sized card, which they gave to study personnel at each follow-up visit.Semen analyses methods for both studies were based on World Health Organization recommendations, but the methods differed somewhat between the two studies. Freshly collected semen was examined in the fascial interposition study, and data were obtained on sperm concentration, motility, and viability. For the cautery study, two of the four sites did not routinely collect fresh specimens, so semen analysis data from those two sites were limited to sperm concentrations. Therefore, for this comparative analysis, we did not consider sperm motility as an outcome measure. In addition, specimens showing azoospermia or very low sperm concentrations were centrifuged in the fascial interposition study but not in the cautery study. During both studies, the laboratories conducted periodic quality-control tests.In both studies, we used frequent semen analyses rather than pregnancy as the vasectomy effectiveness outcome measure, to minimize the risk of pregnancy, sample size, and study duration. Vasectomy success is commonly defined as two azoospermic specimens . HoweverConsequently, we used two definitions for vasectomy success for this comparative analysis. Our primary definition of success was severe oligozoospermia, defined as <100,000 sperm/mL in two consecutive specimens taken at least two weeks apart. Our alternate definition of success was the occurrence of two consecutive azoospermic specimens taken at least two weeks apart, with no subsequent samples showing sperm concentrations of 100,000 sperm/mL or higher. Motility was not considered, for reasons mentioned earlier. The date of success was the date of the first of the two oligozoospermic or azoospermic semen samples.For early failure, we used a criterion of >10 million sperm/mL at week 12 or later, regardless of motility. This is an adaptation of Alderman's criteria specifying 5 million motile sperm/mL or more as evidence of "overt failure" [The data collection forms, study monitoring, and laboratory quality-control procedures were similar for both studies, though only one research site was common to both. Both studies were organized and managed by researchers and staff at FHI and EngenderHealth, and both received approval from FHI's institutional review board and from institutional review boards at the study sites.Kaplan-Meier product-limit estimates of the probabilities of severe oligozoospermia, at each scheduled week of follow-up through week 24, and their 95% confidence intervals (CIs) were produced overall, by study group , and by study group and age group (i.e., <35 years and 35 years and older). Peto's standard error  was usedThe comparison of failure rates between the two study groups was based on a Fisher exact test with a two-sided alternative hypothesis and an alpha of 0.05. In addition, a logistic model was fit to estimate an age-adjusted odds ratio of the failure rates and its 95% CI.Unlike in the cautery study, participants in the fascial interposition study were discontinued after azoospermia was confirmed or after vasectomy failure was declared. Therefore, for the purpose of comparing the distribution of the participants in the different sperm concentration categories by week of follow-up, we kept azoospermic cases in the azoospermic category for all follow-up weeks after their discontinuation due to confirmed azoospermia. Similarly, we kept participants with a declared vasectomy failure in the sperm concentration category that they were in at the moment of discontinuation, for all subsequent follow-up weeks.Detailed results for the two studies have been reported ,6. We reAmong the baseline population characteristics Table , age disWe found significantly fewer early failures in the cautery study than in the fascial interposition group from the randomized controlled trial: 1.0% (4/389) versus 4.9% (20/410) (p = 0.0014 by the Fisher exact test). The adjusted odds ratio was 4.8 (95% CI, 1.6–14.3), indicating nearly a five-fold higher risk of early failure in the fascial interposition study than in the cautery study. No significant age effect was detected (data not shown).The distribution of sperm concentrations by week is shown for the two studies Figure . The difLife-table analyses of time to success showed that the participants in the cautery study reached severe oligozoospermia significantly more rapidly than did those in the fascial interposition study (p = 0.0049) Figure . Ninety-The difference in the observed failure rates suggests that vas occlusion techniques that include cautery are significantly more effective than ligation and excision plus fascial interposition, at least based on semen analysis. We believe that most of the failures in the fascial interposition group were due to early recanalizations within the first two to three months after vasectomy (data not shown). Possible explanations for the surprisingly high failure rate among the fascial interposition group have been previously presented and discussed .Recent reports suggest that pregnancy rates may be higher in low-resource settings, where semen analysis is usually not available and where most vasectomies are done by ligation and excision ,13. The When a ligation and excision technique is used, we have shown that fascial interposition provides an important improvement in effectiveness . The addSchmidt was a pioneer in the use of cautery for vasectomy. His preferred technique was thermal cautery of about 5 mm of each end of the vas, combined with fascial interposition and no excision . Two of The length of the vas segment that is cauterized can vary by the type of cautery. In the United States and Canada, marketed thermal cautery "vasectomy tips" have a functional length of about 0.8 cm, so it would be difficult to cauterize more than 1 cm of each end. The cautery tip of the Sturgeon cautery device used by Schmidt was 0.5 cm long. However, the tips available for electro-cautery devices are much longer, which could permit cauterization of longer segments of the vas and potentially cause more difficulty in the event of a request for reversal.Since several reports suggest that the combination of thermal cautery plus fascial interposition is one of the most effective methods available , this prSchmidt suggested that cautery should not be combined with suture or clip ligation of the vas . He noteLimitations of this comparison include (a) the variety of vas occlusion techniques used in the cautery study and differences in methods of fascial interposition between the two studies, (b) the uncertain correlation between post-vasectomy sperm concentrations and the risk of pregnancy, (c) the lack of sperm motility data and centrifugation for the cautery study, and (d) the use of different study sites and surgeons in the two studies. The difference in time to vasectomy success could in part be related to the lack of centrifugation in the cautery study. However, the difference in centrifugation between the two studies would not have affected the detection of early failures, especially since the cautery study gathered semen samples throughout the 24-week follow-up period (i.e., men continued providing semen samples even after they reached azoospermia). Another potential limitation of this comparison was the difference in follow-up schedules between the two studies.In addition, other factors could have an unknown impact on the comparability of the data. Even though the results are encouraging for the use of cautery in vasectomy, we have to be cautious about making definitive statements based on this nonrandomized comparison.Results from this comparative analysis suggest that cautery may be a more robust and less technique-dependent method than is fascial interposition. However, additional research would be useful to directly compare the effectiveness of the following standardized techniques in a randomized controlled trial: cautery without fascial interposition, cautery with fascial interposition, and ligation and excision with fascial interposition. Additional research would also be of interest to compare an open-ended procedure with a closed-end procedure. Several investigators have suggested that leaving the testicular end open reduces post-vasectomy pain, but no randomized controlled trials have examined this issue ,18.We compared data from two prospective multicenter studies conducted using similar methodologies. We found that the use of cautery as part of the vasectomy procedure significantly reduced vasectomy failure rates compared with ligation and excision plus fascial interposition as part of the procedure. It is unclear from our results and those of others whether fascial interposition used with cautery improves vasectomy success rates when compared with cautery alone.The authors declare that they have no competing interests.DS participated in the conception, design and analysis of the study, and drafted the manuscript. BI participated in the conception, design, and analysis of the study, and was primarily responsible for managing the study implementation. MC participated in the design of the study and was primarily responsible for the statistical analysis. ML participated in the conception, design and analysis of the study. MB participated in the conception, design, management and analysis. All authors reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Plasmodium seasonal behaviour is reviewed and three historical data sets, concerning seasonal transmission of Plasmodium falciparum, are examined. It is proposed that the dramatic seasonal increase in the density of uninfected mosquito bites results in an increased infectiousness of the human reservoir of infection and, therefore, plays a key role in "kick-starting" malaria parasite transmission.The regional malaria epidemics of the early 1900s provided the basis for much of our current understanding of malaria epidemiology. Colonel Gill, an eminent malariologist of that time, suggested that the explosive nature of the regional epidemics was due to a sudden increased infectiousness of the adult population. His pertinent observations underlying this suggestion have, however, gone unheeded. Here, the literature on  Plasmodium [A strategic aim of the global commitment to Roll Back Malaria is the development of reliable and practicable forecasting methods to enable the containment of epidemics. Much of the basis of our knowledge about the causes and dynamics of malaria epidemics comes from the qualitative analyses of the large scale, regional epidemics occurring in the first third of the last century -3. Such asmodium .Plasmodium vivax and Plasmodium falciparum) in the adult population. If Gill was indeed correct, then what caused the sudden increase in infectiousness of the adult population?Despite the extension by Macdonald of the basic models of malaria, pioneered by Ross and Lotka, to introduce some level of biological complexity, many points raised at the time of the great regional epidemics remain unexplored or attributed to the error inherent in estimating biological parameters . In his Plasmodium  has a very high R0 (reproductive rate) that is strongly dependent on the mosquito biting rate. But where do the initial source infections come from? Several authors have noted the absence of gametocytes during inter-epidemic periods, but commented that a geometric rise in gametocyte carriers could generate the observed rapid increase in gametocyte rates during the course of an epidemic [P. falciparum infection can produce infective gametocytes throughout its infection duration [Plasmodium spp. (notably P. vivax), is well known and yet rarely considered in models of malaria epidemiology. In this paper, the nature of seasonality in malaria parasites is addressed and by examination of three historical data sets from Africa and Asia, it is proposed that uninfected mosquito bites play a significant role in increasing the infectiousness of the human reservoir of infection to mosquitoes. Thus, the intense expansion of the mosquito population at the start of the seasonal transmission season (whether resulting in an epidemic or not) has a significant biological effect above and beyond the role of vector.Seasonal epidemic rises superimposed on endemic prevalence are probably characteristic of most regions with endemic malaria ; there iepidemic -16. In aepidemic ,18. Howeduration . Even thduration , which sP. vivax in temperate regions is legendary, and was discussed in depth by Swellengrebel and De Buck (1938) [Anopheles. Korteweg proposed that such fevers were the result of infections incubating from the previous year. Induced infection studies, implementing P. vivax and P. falciparum to treat neurosyphylitic patients, by Swellengrebel and others confirmed Korteweg's hypothesis that vernal fevers were due (at least in part) to latent vivax infections from the previous autumn [The vernal (late winter – early spring) rise in the incidence of k (1938) . Such ves autumn -22. A vas autumn , using ts autumn , using tP. vivax spring relapses was a tendency for gametocyte production just before mosquitoes appeared. This is a feature of other haemosporidian parasites, most notably Leucocytozoan and Haemoproteus spp. that are widespread parasites of birds and lizards, the latter of which have recently been shown to be paraphyletic with Plasmodium species [Plasmodium spp. in maintaining the infection exclusively by exo-erythrocytic forms and only gametocytes are present in the circulating blood system; seasonal activity and the timing of gametocyte production are thus simultaneous in these genera. There is a rapid appearance of gametocytes in March and April, which is generally too early for novel infections by simulid black flies or ceratopogonid midges [Haemoproteus infections normally relapse during host breeding, peaking during egg laying and decreasing throughout the nestling rearing phase [Plasmodium infections in lizard malaria species. Gametocyte production is intensified during mid-late summer in Plasmodium mexicanum infections in lizards when similar seasonal fluctuations occur in testosterone levels. Although testosterone does not seem to affect overall parasite density and the course of infection, elevated testosterone significantly reduced variation in the timing of the onset of gametocyte production [Transmission to mosquitoes is achieved through the production of gametocytes. Ziemann (1914)  noted th species . These pd midges ; rather ng phase . This reng phase . An infloduction .P. vivax and Hepatocystis spp.  in the tropics is less evident and thought not to occur [P. falciparum be detected? As stated previously, current wisdom holds that there is a continual production of low numbers of gametocytes that are the source of infection once mosquito numbers expand. However, contrary to accepted belief, mosquitoes can be found at all times of the year, even under very hostile climatic conditions [In contrast to temperate and subtropical zones, seasonal patterns of parasite relapse in parasites such as to occur . Unlike nditions  and seasnditions  is consinditions  summarizPlasmodium spp. would have evolved to respond to the mosquitoes themselves. Thus, in a manner akin to temperate parasite species that utilize vertebrate host seasonal cues to produce gametocytes at the optimal time, tropical species may respond to tropical cues – the mosquito bites. Is there any evidence that P. falciparum, for example, produces gametocytes in response to mosquito bites? As with P. vivax, P. falciparum gametocyte production has been found to peak at a time when the anopheles abundance was at a maximum [P. falciparum transmission, three historical data sets, addressing age-specific longitudinal patterns of P. falciparum asexual and sexual prevalence rates in relation to mosquito abundance patterns, are analyzed. One of the major shortcomings of these studies is that they did not have recourse to PCR (quantitative, reverse transcriptase) technology and thus will have underestimated parasite prevalence rates and can not distinguish a relapsed chronic infection from a novel infection. However, despite such limitations, the data sets reveal much on the seasonality of P. falciparum transmission.Such anopheline data are one side of the coin and gametocytes are the other. If tropical seasons are defined most precisely by mosquito bionomics, it is conceivable that  maximum -35. Howe maximum . The hum maximum ,37,38, i maximum ,38-40. C maximum -41. More maximum ,43; cons maximum -46 than P. falciparum seasonal prevalence data. During the same period, intensive surveys of anopheline activity and mosquito infection prevalence rate  infection rates) were carried out throughout Lagos by indoor resting catches in from 200 to more than 400 rooms. Infants (three to four months) are no longer expected to be protected by maternal antibodies and prevalence rates in this very young "naïve" age group provide a good indication of the force of infection and hence the current transmission intensity. Mothers will include a distribution of older age groups that are expected to have developed some immunity to infection and disease. It should be noted that although Plasmodium infection characteristics in pregnant and recently post-partum women differ from non-gravid women of similar ages [For a period of 18 months, records of vaccine visits to the Lagos local health office of 3–4 month old babies with their mothers provided lar ages ,49, the Using the mosquito data, the estimated monthly entomological inoculation rate (EIR) (mosquito density × mosquito biting rate × sporozoite rate) and the estimated human reservoir of infection (infectious gametocyte rate) are calculated, using the formula of Macdonald (1952) . where s (sporozoite rate),  (infective mosquito lifespan), a  and x is the proportion of human infections that are infectious to mosquitoes .p can be calculated from the measured total mosquito infection rates: sporozoite rate, s, and zygote rate, z.n and m are the number of days required for the development of identifiable presence of sporozoites and oocysts respectively (i.e. the extrinsic incubation periods for sporozoites and oocysts). Here standard values of n = 12 and m = 3 [ and a mean annual temperature T fluctuating around 25°C.where nd m = 3 ,50 were x can be estimated, thereby enabling examination of how the infection rates in the two directions, mosquito  man (EIR) and man  mosquito (infectious gametocyte rates), relate to the age-specific (three to four month old infants versus mothers) seasonal changes in parasite prevalence rates and the mosquito activity patterns , there is a parallel increase in prevalence of infection in both the young and older age groups Fig. , a largeIn the final "End" phase, prevalence rates drop markedly and notably more rapidly in the younger age class Fig. , indicatPeak and End phases follow the classic description of a malaria transmission season, most especially confirming the sensitivity of the infant versus the adult age groups in revealing changes in the transmission intensity and the relationship between transmission and the (estimated) patterns of gametocyte dynamics. However, the Start phase highlights a novel effect whereby there is little effective transmission (infant rates or estimated gametocyte rates) but an increase in adult prevalence rates, which notably parallels the dramatic increase in overall biting rate, predominantly by uninfected anophelines. This pattern suggests that the increase in mosquito activity per se may be having an effect on parasite prevalence rates in the adult population. In high endemicity areas, subpatent infections are common and likely to occur in older age groups with a previous history of exposure [P. falciparum in highly endemic areas, which have demonstrated age dependence in both the multiplicity of infection and the relationships between this multiplicity and the risk of acute illness: in older children, a high multiplicity of infection is characteristic of low-level chronic parasitaemia [per se cause the re-emergence of existing infections in this adult population.The data in the exposure . This cositaemia ,53. In asitaemia . Althougsitaemia  may be sThis hypothesis is to some extent corroborated by observed patterns of parasite prevalence versus parasite density in an adult population in Liberia . Here, aAnopheles albimanus productivity from larval collections was at a maximum at the very time when sudden cold spells were producing a wave of relapses and a crop of gametocyte carriers. At that critical period, at the beginning of the malaria season, the increase in gametocyte rate was particularly marked in the group >7 years of age. In this age group, the gametocyte rate increases from 1.1% in the summer to 17.3% in the rainy season. Muirhead-Thomson [P. falciparum infections positive for gametocytes varied little with season (from 67 to 62%), whereas in the older age group the change was marked, increasing from 22 to 61%. Why should the two groups display very different changes in gametocyte production but similar increases in prevalence rates? Could mosquito biting be having an effect on infections in the older age group?Such age-specific effects have also been noted by Muirhead-Thomson  during l-Thomson  suggesteThe second data set comes from a longitudinal study in a rural village in Tanzania characterised by seasonally intense transmission. In this study both gametocyte and asexual prevalence rates were measured intermittently (every two or three months) and classified into three age groups : <5 years, 6–20 years and 20+ years. In addition, anopheline activity and sporozoite rates were measured on a monthly basis. Wilson noted distinct seasonal fluctuations in gametocyte rates, whereas overall parasite prevalence rates varied little. The longitudinal age-specific parasite and gametocyte prevalence rates are shown in Fig. per se on parasite prevalence rates in the adult population.The parasite prevalence rates change very little but do generally follow the monthly fluctuations in EIR; the absence of monthly data points, however, abnegates any rigorous comparison. The age-specific prevalence rates are characteristic of hyperendemic transmission intensity with peak rates in the very youngest age group. As discussed in the previous study , the occet al. (1990) [P. falciparum and P. vivax malaria in S.E. Thailand. During the two year study period, monthly human and mosquito data were collected. Despite the very different nature of malaria epidemiology in Thailand, when compared with the previous two African studies, Rosenberg et al. (1990) [et al. (1990) [et al. [vs. 8x higher in the 14+ age group ; (iii) in year two, the EIR decreased by 67% and, whereas the gametocyte prevalence decreased in <14 age group, it increased in the 14+ group.Rosenberg . (1990) -60 condu. (1990)  – « None. (1990) . In addiThe younger age groups have higher gametocyte densities reflecting their generally higher parasite loads. Younger age groups also tend to have higher rates of acquisition and recovery (i.e. turnover) and, therefore, most gametocytes would be expected to be due to novel infections. This is supported by the finding that gametocyte prevalence decreased in the <14 group in year 2 when inoculation rate and force of infection was less. Gametocyte prevalence increased in the 14+ group despite a decrease in inoculation rate in year two; note, however, that despite the drop in EIR, the mosquito biting intensity was higher. Thus, the apparent paradox of there being considerable novel infections at the beginning of the transmission season, but with no influence on parasite prevalence, may be explained by the re-emergence of pre-existing chronic infections in the 14+ age group. This group is more likely to harbour such infections and is shown here to display the most significant seasonal changes in gametocyte rates .P. falciparum trophozoite density infections, suggesting an absence of novel infections, there is a small rise in gametocyte rates. The following month (December), this increased EIR leads to a dramatic increase in high P. falciparum trophozoite density infections and gametocyte prevalence rates. A similar pattern is seen in the second year, and notably, although the EIR and sporozoite rates were lower, resulting in reduced gametocyte prevalence rates in children, the peak mosquito biting density (which again preceded the increase in sporozoite rate) was higher, as was the gametocyte rate in adults. Whereas the large concomitant increases in high P. falciparum trophozoite density infections and gametocyte prevalence rates can be taken as evidence for novel/super infections, the increase in gametocyte rates in the absence of sporozoites at the start of transmission, coupled with the variation in age-specific gametocyte rates between the two years, suggests once more that mosquito biting itself may induce gametocyte production and subsequently augment mosquito infection rates.The sequence of events in this study could thus be interpreted as follows : In year one, the mosquito biting rate increases dramatically in October, with a concomitant monthly rise in oocyst rates, but not sporozoites (data not shown here), and no rise in gametocyte rates. In November, the sporozoite rate and EIR increase as expected from the previous month's oocyst rates and the decrease in newly emerged mosquitoes (evident from the parous rate ). DespitAedes spp.), for example, have a capacity to increase more rapidly in numbers than anophelines; this is because of the ability of their eggs to resist dessication. Thus, the seed population of culicines is probably greater than that of anophelines, which must expand from pockets of aestivating females. Clearly this view is very preliminary, but the available information is encouraging and this hypothesis warrants closer examination in regions of endemic seasonal transmission.Here, a case is presented for there being a role of uninfected mosquito bites in increasing the human reservoir of infection at the beginning of the transmission season. The available data is limited, but consistently suggest that the traditonal view that there is a rapid expansion of the parasite population from a few source infections following the seasonal increase in anophelines is overly simplistic. It must, however, be emphasised that in no way is it suggested that once transmission is under way, the epidemiology of malaria is not satisfactorily described by classic Ross-Macdonald models or variants thereof . Rather,If, indeed, there is a role for mosquito bites, this could have far reaching possibilities, not only for novel intervention strategies, but also for strategies in combatting malaria in regions of seasonally intense transmission, including regions at risk of epidemics. If intervention measures can be taken to reduce the potential reservoir of infection, by targeted use of antimalarial drugs with gametocytocidal effects during the window of mosquito resurgence, for example, the increase in transmission intensity will be severely delayed. Moreover, reducing human-mosquito contact by the use of insecticide treated bednets (ITBNs) or repellents may have considerable impact at this crucial time. A mass action community level benefit of ITBNs has already been noted, where individuals not sleeping under nets in villages where bednets were implemented, had reduced mortality and morbidity rates ,65. ITBNRELP, MD and PTB were all involved in the conception of the article that arose out of lengthy discussions among the authors. The final draft was written by RELP with various sections added and/or removed by MD and PTB.


B receptors in the ventral tegmental area (VTA) has been suggested to attenuate the rewarding properties of psychostimulants, including nicotine. However, the neurochemical mechanism that underlie this effect remains unknown. Since GABAB receptors modulate the release of several neurotransmitters in the mammalian brain, we have characterised the effect of the GABAB receptor agonist baclofen on the release of [3H]-dopamine ([3H]-DA) from VTA slices of naïve rats and of rats pre-treated with nicotine.The activation of GABA3H]-DA from the isolated VTA (EC50 = 0.103 μM, 95% CI = 0.043–0.249), without affecting the basal [3H]-monoamine overflow. This effect was mediated by activation of GABAB receptors as it was blocked by the selective receptor antagonist CGP55845A. Chronic administration of nicotine (0.4 mg kg-1, s.c., for 14 days) affected neither the basal nor the electrically evoked release of [3H]-DA from VTA slices. However, the inhibitory effect of baclofen (10 μM) on the stimulated [3H]-monoamine overflow was abolished in rats pre-treated with nicotine as compared to saline-injected controls.In naïve rats, baclofen concentration-dependently inhibited the electrically evoked release of [B receptor activation reduces the release of DA from the rat VTA. In addition, a reduced sensitivity of VTA GABAB receptors appears to develop after chronic exposure to nicotine. The resulting disinhibition of VTA DA neurones might therefore contribute to the sensitised dopaminergic responses observed in the rat mesocorticolimbic system following repeated administration of nicotine.Our results demonstrate that GABA The ventral tegmental area (VTA) represents the site of origin of the mesocorticolimbic dopaminergic pathway that has been implicated in mediating the reinforcing properties of drugs of abuse, including nicotine -3.The majority of cells within the ventral tegmental area consist of dopaminergic, tyrosine-hydroxylase containing neurones, which send axon projections to forebrain structures such as the nucleus accumbens and the prefrontal cortex. The non-tyrosine hydroxylase containing neurones are mainly GABAergic and function either as local interneurones to modulate the activity of the principal dopaminergic cells or as projection neurones providing inhibitory input to the cortex and the nucleus accumbens -8.2 autoreceptors inhibits excitability and firing rate of VTA dopaminergic neurones [1 receptors, which are found on GABA- and excitatory amino acids-containing terminals in the VTA [In addition to classical release from the axon terminals located in the forebrain, midbrain dopaminergic neurones release dopamine (DA) from their soma and dendrites -12. The neurones ,14 and dneurones ,16. Furt the VTA -20.A and GABAB receptors, respectively [B receptors has been reported to inhibit the spontaneous pacemaker-like activity of VTA DA neurones in slice preparations [in vivo [Besides the short-loop feedback inhibition exerted by dopamine, the activity of VTA dopaminergic neurones is strongly modulated by glutamatergic and GABAergic inputs. Activation of NMDA receptors by excitatory afferents arising from the medial prefrontal cortex ,22 inducectively . Activatarations ,28,29 an[in vivo ,31.in vivo microdialysis studies have demonstrated that intra-VTA administration of the GABAB receptor agonist baclofen decreases extracellular dopamine levels in both the somatodendritic [B receptor-mediated inhibition of the activity of the mesocorticolimbic neurones might explain the effectiveness of baclofen to suppress nicotine self-administration when microinjected into the VTA [In addition, endritic ,33 and tendritic ,16. The  the VTA . In fact the VTA ,35. Acut the VTA . Sensiti the VTA -39.B receptors have a prominent role in regulating the activity of VTA DA neurones and they appear to be involved in the modulation of nicotine reinforcing properties, we have characterised the effect GABAB receptor stimulation on the somatodendritic release of [3H]-DA from VTA slices of naïve rats and used this model to determine whether chronic exposure to nicotine results in altered GABAB receptor-mediated modulation of VTA DA cells.Since GABA3H]-DA from ventral tegmental slices of naïve rats was evaluated in order to determine its physiological significance under the experimental conditions used here. Removal of calcium with the inclusion of EGTA (1 mM), or addition of TTX (1 μM) to the superfusion buffer abolished the stimulated release of [3H]-DA from the tissue, without significantly affecting the spontaneous monoamine overflow. Recovery of the release occurred during the second stimulation (S2), after removal of TTX or replacement of calcium to the superfusion buffer  and calcium on the electrically evoked release of [ulation S, after r3H]-DA release from VTA slices , added to the superfusion buffer 36 min before the second stimulation, dose-dependently reduced the electrically evoked [3H]-DA was abolished when the selective GABAB receptor antagonist CGP55845A (1 μM), which had no effect on its own, was added to the superfusion buffer concomitantly with baclofen ; 0.99 ± 0.13 for CGP55845A alone; and 0.93 ± 0.14 for slices perfused with both baclofen and CGP55845A.The inhibitory effect of baclofen (10 μM) on the release of [3H]-DA from the VTA slices of rats that received a chronic nicotine treatment was compared with the overflow observed in saline-control animals. Neither the basal nor the electrically stimulated release of [3H]-DA was significantly affected by the drug pre-treatment . By contrast, in the rats pre-treated with nicotine, the addition of baclofen to the superfusion buffer did not produce any significant effect on the evoked release of [3H]-DA  . Taken together, our findings suggest that after chronic nicotine the GABAB receptor may be desensitised, and this would result in a reduced inhibitory control of VTA dopaminergic cells, thereby facilitating a more sustained increase in the responses of mesolimbic neurones to nicotine. Interestingly, the inhibitory action of baclofen on VTA DA cells may be accounted for by stimulation of GABAB receptors located on dopaminergic and/or glutamatergic neurones [B receptors located either on DA cell bodies or, presynaptically, on glutamatergic terminals impinging onto DA neurones. The latter hypothesis would lead to the speculation that a chronic treatment with nicotine might result in increased excitatory input to the VTA, due to a reduced GABAB-mediated inhibitory control. Therefore, disinhibition or increased excitatory input to midbrain DA cells might contribute to the augmented dopamine output observed in the nucleus accumbens after a challenge injection of nicotine in rats pretreated with the drug [B receptors located in the VTA has also been reported to occur after chronic cocaine administration, as the drug treatment resulted in reduced functional coupling of the receptor to G-proteins [B receptor expression and coupling to G-proteins in the ventral tegmental area of the rat are not altered after chronic exposure to nicotine [in vitro autoradiography did not allow us to discriminate between receptor subpopulations, including receptor subtypes involved in different functions. Thus, if the GABAB receptors directly implicated in the control of dopamine release from the VTA represent only a small fraction of the overall receptor population in this area, autoradiographic analysis would not be sufficiently sensitive to evaluate receptor modifications occurring after chronic exposure to nicotine. Further studies aimed at characterising the mechanisms involved in GABAB receptor desensitisation following chronic nicotine treatment are required to clarify this issue.Interestingly, previous neurochemical studies have shown that activation of GABAccumbens  and the ccumbens . The redccumbens . Howeverneurones ,29. Thisthe drug ,37. Deseproteins . Howevernicotine , suggest3H]-DA from the in vitro preparation, no difference was found in the K+-evoked release between the drug pretreated animals and the saline-injected controls [B receptor in the ventral tegmental area of rats pretreated with nicotine.Not surprisingly, basal and evoked release of dopamine from VTA slices of rats chronically injected with nicotine did not differ from release obtained from saline-control tissue. This confirms that sensitised dopaminergic responses to the drug depend on the activation of nicotinic receptors (nAChRs) and are not the result of a generic increase in neuronal activity. Similar results have been obtained in striatal synaptosomes from rats pretreated with the nAChR agonist anatoxin-a for 7 days: while the drug pre-exposure increased the nicotine-stimulated release of [controls . Thus, aB receptors inhibits the release of preloaded [3H]-DA from somatodendritic fields of VTA neurones in naïve rats. This effect appears to be reduced in rats chronically treated with nicotine, suggesting that subsensitivity of GABAB receptors in the VTA might occur as a result of the drug treatment. This, in turn, would lead to disinhibition of VTA dopaminergic cells, which might contribute to the increased activity of mesocorticolimbic neurones following repeated exposure to nicotine.In conclusion, we have demonstrated that activation of GABA3H]-Dopamine (specific activity 47 Ci/mmol) was obtained from Amersham (Buckinghamshire, UK). (-)-Baclofen (CGP11973A) and CGP55845A were generous gifts from Novartis Pharma (Basel, Switzerland). (-)-Nicotine hydrogen tartrate salt, nomifensine, pargyline, tetrodotoxin and ethylene glycol-bis (b-amino ethyl ether) tetraacetic acid (EGTA) were purchased from Sigma-Aldrich (Dorset, UK). All the other chemicals used in this study were obtained from Fisher Scientific (Leicestershire, UK).[Male Wistar rats (weight 250–280 g) were maintained on a 12-h light/dark schedule (on 6:00–18:00), with free access to food and water.-1, expressed as free-base) or vehicle (0.9% NaCl, 1 ml kg-1) for 14 consecutive days, once a day, between 9:30 and 10:30 a.m. Experiments were performed 24 hours after the last injection. All procedures were carried out in accordance to the UK Animals (Scientific Procedures) Act, 1986.For the drug treatments, rats, initially weighing 150–180 g, received subcutaneous injections of nicotine , saturated with 95% O2/5% CO2.Animals were sacrificed by stunning followed by decapitation. The brains were rapidly removed from the skull and cooled for 2–3 min in ice-cold superfusion buffer of the following composition (in mM): NaCl, 118; KCl, 4.7; CaClEach brain was dissected according to visual anatomical landmarks and the atlas of Paxinos & Watson . To obtaThe dissected VTA was cross-chopped (250 μm × 250 μm) with a McIlwain tissue chopper and the tissue slices were washed and resuspended in ice-cold superfusion buffer.3H]-DA for 30 min, in the dark and in the presence of 10 μM pargyline. The incubation was terminated by washing the slices three times with buffer containing 2.5 μM nomifensine.The VTA slices were pre-incubated in oxygenated superfusion medium at 37°C for 15 min, followed by incubation with 0.1 μM [-1 with oxygenated buffer maintained at 35°C and containing 2.5 μM nomifensine, to block dopamine uptake, and 10 μM pargyline to ensure that [3H] overflow represented primarily [3H]-DA rather than its metabolites [150 μl aliquots of slice suspension (1.5 to 2.0 mg of tissue) were transferred to each chamber of a Brandel 2000 superfusion apparatus. The tissue was superfused at a rate of 0.5 ml minabolites .3H]-DA was induced by electrical field stimulation (20 mA, 2 Hz for 4 min) using a Brandel constant current stimulator. Two stimulations occurred 14 min (S1) and 59 min (S2) after the beginning of sample collection.After 36-min pre-superfusion, the effluent was collected in consecutive fractions of 4 min 30 sec each. The release of [2+-free buffer (in the presence of 1 mM EGTA), or buffer containing 1 μM TTX, for 15 min before and during S1. The superfusion medium was then replaced with normal buffer until the end of superfusion and during the second stimulation.Test drugs were added to the superfusion medium 36 min before the second stimulation. To determine the effects of calcium and tetrodotoxin (TTX), slices were perfused with Ca® 1500 liquid scintillation analyser (Packard Bioscience Company), programmed to count for tritium, 3 minutes per vial, at an efficiency of 60%. The number of disintegrations per minute (d.p.m.) was measured in order to determine the concentration of tritium in each sample.At the end of the superfusion, the slices with their filters were removed from the chamber, suspended in 1 ml of buffer, and sonicated. OptiPhase 'HiSafe' 3 scintillation fluid (Perkin Elmer, UK) was added to each vial and the radioactivity content in the superfusion samples and in the tissue slices was assayed by liquid scintillation counting using a Tri-Carbi.e., as a percentage of the amount of radioactivity in the tissue at the beginning of that collection. The electrically evoked release was expressed as the mean of the increased fractional release above baseline in the two fractions after the beginning of stimulations. Basal release, in turn, was calculated as the mean of the amount of radioactivity present in the three samples just before each period of electrical stimulation.For each time point (4 min 30 sec), release of radioactivity was expressed as fractional release, n independent experiments conducted in either triplicate or quadruplicate. For statistical analysis one-way ANOVA with Dunnett's post hoc test was used to compare values of S2/S1 in the presence of a drug versus values of S2/S1 from control slices superfused with buffer alone. When nicotine or saline were administered to the animals, values of S2/S1 were analysed by ANOVA with repeated measures (with or without baclofen) with pre-treatment as factor analysed, and post hoc comparisons were made using the Bonferroni test. The accepted level of significance was p < 0.05.Results were expressed as mean ± s.e.mean of 2O2) for 30 min, followed by incubation with 0.2% Triton X-100 (Sigma-Aldrich) for 20 min at room temperature.Immunohistochemistry was performed on naïve rat brain during preliminary experiments aimed at characterising the exact orientation of the cutting for the dissection of the VTA Figure . AnimalsAfter a 1-hour pre-incubation in 10% normal goat serum (NGS, Vector), the primary antibody  was applied at a final concentration of 1:1000 (in 0.01 M PBS) and the sections were allowed to incubate overnight at 4°C. After three washes with fresh buffer, the slices were incubated for 90 minutes at room temperature with a biotinylated secondary goat anti-rabbit antibody (1:200 dilution, Chemicon).Immunoreactivity was visualised by the avidin-biotin complex method of detection (Vectastain Elite ABC Kit, Vector) using 3,3'diaminobenzidine (DAB, peroxidase substrate kit, Vector) as the chromogen.DA carried out the experiments, participated in the design of the study and drafted the manuscript.NGB conceived the study, and participated in its design and coordination.All authors read and approved the final manuscript.


Undifferentiated Nasopharyngeal Carcinoma (NPC) patients show a characteristic pattern of antibody responses to the Epstein-Barr virus (EBV) which is regularly associated with this tumor. However, no EBV-specific cytotoxic activity is detectable by the standard chromium-release assay at both peripheral and intratumoral levels. The mechanisms underlying this discrepancy between the humoral and cellular immune responses in NPC are still unknown, but might be related to an imbalance in immunoregulatory interleukin production. In this report, we investigated the ability of peripheral (PBL) and tumor- infiltrating (TIL) lymphocytes of undifferentiated NPC patients to produce in vitro three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA).Lymphocytes from 17 patients and 17 controls were cultured in the presence of Pokeweed mitogen (PWM) for 12 days and their culture supernatants were tested for interleukins and immunoglobulins by specific enzyme-linked immunosorbent assays (ELISA). Data were analysed using Student's t-test and probability values below 5% were considered significant.The data obtained indicated that TIL of NPC patients produced significantly more IL-2 (p = 0,0002), IL-10 (p = 0,020), IgM (p= 0,0003) and IgG (p < 0,0001) than their PBL. On the other hand, patients PBL produced significantly higher levels of IL-2 (p = 0,022), IL-10 (p = 0,016) and IgM (p = 0,004) than those of controls. No significant differences for IL-6 and IgA were observed.Taken together, our data reinforce the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. An increased ability to produce cytokines such as IL-10 may underlie the discrepancy between humoral and cellular immune responses characteristic of NPC. Undifferentiated nasopharyngeal carcinoma (NPC) is a malignant epithelial tumor characterized by a heavy infiltration of non malignant lymphocytes and most of these tumor infiltrating lymphocytes (TIL) have been shown to be T cells .The Epstein-Barr virus (EBV) is causally associated with this malignancy since viral DNA is regularly present in the malignant epithelial cells but not in the neighbouring normal tissues. In addition, NPC patients show a specific pattern of humoral responses against EBV antigens .Viral proteins known to be expressed in NPC tumor cells are the EBV-encoded nuclear antigen 1 (EBNA-1) and the latent membrane proteins LMP-1 in 35 to 65% of cases, and LMP-2 . The latRecently, CD8 positive EBV-specific cytotoxic T cell clones were isolated from the peripheral blood and tumors of NPC patients . The majThe expression of IL-10 in NPC has been controversial. While it has been reported that IL-10 is not expressed by NPC cells as detected by RNA in situ hybridisation , some reIn this report, we investigated the ability of both peripheral blood lymphocytes (PBL) and TIL of undifferentiated NPC patients to express three interleukins (IL-2, IL-6, IL-10) and three immunoglobulin isotypes (IgM, IgG, IgA) following pokeweed mitogen (PWM) stimulation in vitro.The data obtained indicated some significant differences between NPC patients and controls in interleukin and immunoglobulin production. However, further investigations are needed to establish the relevance of these differences to the discrepancy between humoral and cellular immune responses characteristic of NPC.17 untreated Tunisian NPC patients were included in this study. Informed consent was obtained from all patients before collection of blood and biopsy samples. These patients presented for treatment at the National Cancer Institute Salah Azaiez, Tunis or at the Farhat Hached hospital, Sousse, Tunisia. Their mean age was 47,5 years (range 25–70). All patients were diagnosed histopathologically as undifferentiated NPC.17 healthy EBV carriers who presented to donate blood at the National Blood Transfusion Centre, Tunis, Tunisia, were included as controls in this study.Sera of all patients and controls were titrated for antibodies directed against EBV antigens by indirect immunofluorescence .6 cells/ml.Peripheral blood and biopsy samples were taken on the same day from each of the 17 NPC patients. Peripheral blood was collected from patients and healthy donors in heparin. Mononuclear cells were isolated by centrifugation over Ficoll-Hypaque (Pharmacia) according to Boyum . Followi6 cells/ml.For the preparation of tumor-infiltrating lymphocytes, biopsies were collected from NPC tumors under sterile conditions and immediately transferred to RPMI-1640 medium supplemented with 50 μg/ml Gentamicin. The biopsies were washed several times in this medium and then minced into small pieces in order to extract lymphocytes. The extracted cells were then washed by centrifugation in the same medium and then resuspended in complete medium at a final concentration of 2 × 102 fragment. The labelled cells were counted under a fluorescence microscope.Samples from each lymphocyte preparation were taken for immunophenotyping by indirect membrane immunofluorescence using mouse monoclonal antibodies directed against CD3, CD4, CD8 or CD19 and FITC-conjugated goat anti-mouse F(ab')6 cells/ml in complete medium were distributed at 1 ml / well in 24-well Costar plates. One hundred microliters of a predetermined optimal dilution of PWM (Gibco), for treated wells, or complete medium alone, for untreated controls, were added in corresponding wells. The cells were then cultured for 12 days at 37°C, 5% CO2 and 98% relative humidity in a CO2 incubator. At the end of this incubation period, culture supernatants were harvested and used for cytokine and immunoglobulin determination as described below.PBL from healthy donors or NPC patients and TIL from the same patients suspended at 2 × 10Culture supernatants of PBL and TIL stimulated by PWM under optimal conditions were used for both cytokine and immunoglobulin determination by enzyme-linked immunosorbent assays (ELISA).IL-2, IL-6 and IL-10 concentrations were measured using commercial sandwich-type ELISA kits (Immunotech enzyme immunoassays, France) according to the procedures described by the manufacturer.IgM, IgG and IgA concentrations were measured using sandwich-type ELISA assays prepared in our laboratory. Briefly, 96 well-microplates (Greiner, Germany) were coated for 2 hours at 37°C and overnight at 4°C with 150 μl/well of isotype-specific mouse monoclonal antibody (Sigma, France) appropriately diluted in 0,1 M carbonate buffer, pH 9.6. The microplates were washed four times with phosphate-buffered saline pH 7.4 containing 0,1 % Tween 20 (PBS-Tween), then blocked with 300 μl/well of PBS containing 1% FCS for 30 minutes at 37°C. For the assay, 100 μl /well of appropriately diluted culture supernatants in PBS-Tween containing 10% FCS were incubated in coated microplates for 2 hours at 37°C. The microplates were then washed four times with PBS-Tween and 100 μl/well of the appropriate alkaline phosphatase-labeled conjugate (Goat anti-human IgM, IgG or IgA, Sigma-France) were added at a 1:20000 dilution in PBS containing 1% FCS. After a two-hour incubation at 37°C, the microplates were washed and a p-nitrophenylphosphate substrate solution was added. The microplates were incubated for 1 hour at 37°C and the reaction was stopped with 50 μl/well of 1 N NaOH. Immunoglobulin standards were generated using purified human immunoglobulins of each isotype (Sigma, France). The microplates were read at 405 nm on a microplate reader (LP-400, Diagnostic Pasteur). Data were represented as the mean immunoglobulin concentration of triplicate cultures. Immunoglobulin values were obtained by interpolation from standard curves.Data were analysed using "Student's t-test". Probability values below 5% were considered significant.All patients showed a typical serological profile characteristic of NPC as determined by indirect immunofluorescence. Levels of IgG antibodies to VCA were considerably higher than those in healthy donors. Their titers varied from 640 to 2560 (mean titer = 1065), whereas in healthy donors such titers fluctuated between 40 and 160 (mean titer = 70). IgG antibodies to EA were present in NPC patients only, with titers ranging from 20 to 320 (mean titer = 50).Anti-VCA IgA antibodies were detected in all patients  and anti-EA IgA antibodies were detected in only 4 patients . None of the controls showed IgA antibodies against these antigens.Immunophenotypic analysis of the lymphocyte preparations obtained from patients and controls indicated that CD3+ lymphocytes constituted the major subpopulation with a mean frequency of (60 ± 8)% in PBL of both patients and controls and (55 ± 9)% in TIL. CD4+ lymphocytes represented (35 ± 5)% in PBL of both patients and controls and (20 ± 3)% in TIL. CD8+ cells showed mean values of (40 ± 6)% in PBL of patients, (36 ± 4)% in TIL, and (28 ± 5)% in PBL of controls.On the other hand, CD19+ B lymphocytes represented in average (25 ± 5)% in PBL of patients, (23 ± 6)% in TIL, and (22 ± 3)% in PBL of controls.As illustrated in Figures Unstimulated lymphocytes cultured in the absence of PWM did not show any detectable cytokine production (data not shown).The results of immunoglobulin determination showed that the PBL of NPC patients produced significantly higher levels of IgM (8262 ± 5315 ng/ml) than the PBL of controls .Previous reports indicate the absence of EBV-specific cytotoxic T lymphocytes detectable by the standard chromium release assay in both the peripheral and intratumoral compartments -10,21 inThe expression of IL-10 in NPC has been controversial. While some authors have reported that NPC tumor cells do not express IL-10 , others The expression of cytokines such as IL-2 , IL-6 1 and IL-1In this report, we investigated the ability of both peripheral blood and tumor infiltrating lymphocytes of 17 undifferentiated NPC patients to produce cytokines following mitogenic stimulation in culture. Since immunoglobulin isotypes are determined by cytokine patterns, we also looked for possible correlations between measured cytokine levels and immunoglobulin isotypes produced. PWM was chosen to stimulate the lymphocyte cultures because it is known to activate both T and B lymphocytes in humans ,30. In aThe data obtained indicate that the highest levels of IL-2 were produced by TIL cultures followed by patients'PBL. This is in line with a report by Lakhdar et al  showing IL-10, a representative of the Th2 pattern of cytokines, is generally considered immunosuppressive. It inhibits IL-6 secretion by activated macrophages but not by Th2 lymphocytes . In the Cultures of tumor infiltrating lymphocytes secreted significantly higher amounts of IgM and IgG than the PBL of either patients or controls in good agreement with the increased levels of IL-2 and IL-10, since these cytokines are involved in B cell activation and enhance immunoglobulin synthesis ,25. No sIn an attempt to see whether the observed differences in cytokine and immunoglobulin production between patients and controls or between PBL and TIL of patients could be due to differences in the composition of individual lymphocyte preparations, we performed an immunophenotypic analysis of each lymphocyte preparation. As shown in the results, the only significant changes in the proportions of lymphocyte subsets between patients and controls were observed in the CD8 subpopulation which showed an increase in patients PBL and TIL. On the other hand, the CD4 subset showed a significant decrease in TIL. Such differences in lymphocyte subpopulations are not expected to produce the changes in cytokine and immunoglobulin production observed here, since CD4 lymphocytes are well known for being the main source of IL-2 and IL-10, and for their ability to stimulate immunoglobulin synthesis .Recently, CD4+ lymphocyte populations have been shown to be more heterogeneous and complex than previously thought  and new In conclusion, our data point to the possibility of an imbalance in immunoregulatory interleukin production in NPC patients. Their lymphocytes, especially those infiltrating the tumors, showed in particular a high propensity to produce IL-10 following mitogenic stimulation in vitro. Overproduction of this pleiotropic cytokine may lead to a discrepancy between humoral and cellular immune responses similar to that seen in NPC. The relevance of the present observations to the in vivo situation is not yet established and warrants further investigations.The authors declare that they have no competing interests.LFJ designed and carried out the experimental work, performed the statistical analysis and drafted the manuscript. NK maintained the cell cultures and provided technical help. RHK and KB participated in the design and coordination of the study. RK directed the research and finalized the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: