Short communication: Risk factors for sonographically detectable udder edema in overbagged cows at dairy shows.

At dairy cow shows, the appearance of the udder is very important. To show cows at their best, udders are often presented in an overbagged state by extending milking intervals before the show. This practice represents an animal welfare issue because it is associated with behavior changes (e.g., excessive abduction and decreased eating time) and may affect the health of the animal. The aim of the current study was to determine the association between various parameters (e.g., milk yield, days in milk, milking procedures, or administration of nonsteroidal anti-inflammatory drugs, steroids, or diuretics) and the sonographically detectable udder edema score. Data were collected from the show catalogs, by interviewing the exhibitors, by analyzing official veterinary treatment protocols and official milking data, via laboratory analysis of blood samples, and by sonographic examinations of the udders at the show. For sonographically detectable udder edema scoring, 3 scans were taken, 1 on each fore quarter and 1 incorporating both rear quarters at the level of the median suspensory ligament. For grading the scans, a scoring system of 4 grades (score 0 = no edema, 1 = slight edema, 2 = moderate edema, 3 = severe edema) was used. Data from 321 cows of different breeds were collected at 4 highly competitive Swiss dairy shows (shows A-D) between January and September 2017. To determine risk factors for sonographically detectable udder edema, data were analyzed with 2 different logistic regression models. In model 1, the odds ratios were 3.33 (milking intervals of ≥16 h vs. <14 h), 3.84 (show A vs. show C), and 7.39 (dairy breeds vs. dual-purpose breeds). Milking intervals of dairy breeds were significantly higher than those of dual-purpose breeds. In model 2, milking interval was the only significant risk factor with an odds ratio of 9.00 for milking intervals of ≥16 h. Only the milking intervals represented a relevant risk factor in both models; therefore, we concluded that the previously described sonographic udder edema scoring is a useful technique for detecting overbagged udders at dairy shows. Its routine implementation may improve cow welfare at dairy shows.

Alpha-2 macroglobulin in Alzheimer's disease: a marker of neuronal injury through the RCAN1 pathway.

Preclinical changes that precede the onset of symptoms and eventual diagnosis of Alzheimer's disease (AD) are a target for potential preventive interventions. A large body of evidence suggests that inflammation is closely associated with AD pathogenesis and may be a promising target pathway for such interventions. However, little is known about the association between systemic inflammation and preclinical AD pathophysiology. We first examined whether the acute-phase protein, alpha-2 macroglobulin (A2M), a major component of the innate immune system, was associated with cerebrospinal fluid (CSF) markers of neuronal injury in preclinical AD and risk of incident AD in the predictors of cognitive decline among normal individuals (BIOCARD) cohort. We find that A2M concentration in blood is significantly associated with CSF concentrations of the neuronal injury markers, tau and phosphorylated tau, and that higher baseline serum A2M concentration is associated with an almost threefold greater risk of progression to clinical symptoms of AD in men. These findings were replicated in the Alzheimer's Disease Neuroimaging (ADNI) study. Then, utilizing a systems level approach combining large multi-tissue gene expression datasets with mass spectrometry-based proteomic analyses of brain tissue, we identified an A2M gene network that includes regulator of calcineurin (RCAN1), an inhibitor of calcineurin, a well-characterized tau phosphatase. A2M gene and protein expression in the brain were significantly associated with gene and protein expression levels of calcineurin. Collectively these novel findings suggest that A2M is associated with preclinical AD, reflects early neuronal injury in the disease course and may be responsive to tau phosphorylation in the brain through the RCAN1-calcineurin pathway.

Technical note: Evaluation of a sonographic overbagging edema scoring system for show cows: Comparison with visual inspection.

Overbagged udders are commonly seen at dairy cow shows due to prolonged milking intervals. The aims of this study were to describe the prevalence of udder edema in dairy cows at shows as determined by sonography and to suggest a sonographic scoring system, which was evaluated for reproducibility and repeatability. At 4 highly competitive Swiss dairy cow shows, 319 cows of various breeds were examined. Sonographic scans-1 from each fore quarter and 1 from the rear, representing both hind quarters-were collected by 3 experienced veterinarians at defined positions predisposed to overbagging edema. Sonographic scans were scored with the aid of a newly suggested scoring system (score 0 = no edema, grade 1 = slight edema, grade 2 = moderate edema, grade 3 = severe edema). Further, 139 video sequences from behind while the cows were walking and the same number of photographs of the udder each from the left, right, and behind were selected. The photographs and video sequences were visually scored for different parameters as 0 = not present or 1 = present except for abduction, which was scored on a visual analog scale (0 = no abduction; 100 = maximal imaginable abduction). Visual scores were then compared with the sonographic scoring (Pearson's chi-squared). The prevalence of udder edema scores 1, 2, and 3 was 14.0, 6.5, and 2.3%, respectively. Interobserver reliability for objective sonographic scoring (κ = 0.815) and intraobserver agreement (κ = 0.90 and 0.85 for 2 different observers) was estimated as "almost perfect." Interobserver agreements for visual parameters were κ = 0.40 or less except for teat shape (κ = 0.52). Classification of the videos resulted in better interobserver agreement; when rating hind limb abduction, Spearman correlation coefficient was 0.61, whereas κ = 0.61 for absence of udder movement. Udder movement and teat shape were significantly associated with the edema score. The higher inter- and intraobserver reliability of the sonographic edema scoring compared with the visual scoring system indicates that the newly suggested scoring system might be used in the future to objectively identify udder edema in cows at dairy shows.

Midlife adiposity predicts earlier onset of Alzheimer's dementia, neuropathology and presymptomatic cerebral amyloid accumulation.

Understanding how midlife risk factors influence age at onset (AAO) of Alzheimer's disease (AD) may provide clues to delay disease expression. Although midlife adiposity predicts increased incidence of AD, it is unclear whether it affects AAO and severity of Alzheimer's neuropathology. Using a prospective population-based cohort, Baltimore Longitudinal Study of Aging (BLSA), this study aims to examine the relationships between midlife body mass index (BMI) and (1) AAO of AD (2) severity of Alzheimer's neuropathology and (3) fibrillar brain amyloid deposition during aging. We analyzed data on 1394 cognitively normal individuals at baseline (8643 visits; average follow-up interval 13.9 years), among whom 142 participants developed incident AD. In two subsamples of BLSA, 191 participants underwent autopsy and neuropathological assessment, and 75 non-demented individuals underwent brain amyloid imaging. Midlife adiposity was derived from BMI data at 50 years of age. We find that each unit increase in midlife BMI predicts earlier onset of AD by 6.7 months (P=0.013). Higher midlife BMI was associated with greater Braak neurofibrillary but not CERAD (Consortium to Establish a Registry for Alzheimer's Disease) neuritic plaque scores at autopsy overall. Associations between midlife BMI and brain amyloid burden approached statistical significance. Thus, higher midlife BMI was also associated with greater fibrillar amyloid measured by global mean cortical distribution volume ratio (P=0.075) and within the precuneus (left, P=0.061; right, P=0.079). In conclusion, midlife overweight predicts earlier onset of AD and greater burden of Alzheimer's neuropathology. A healthy BMI at midlife may delay the onset of AD.

Using advanced mass spectrometry techniques to fully characterize atmospheric organic carbon: current capabilities and remaining gaps.

Organic compounds in the atmosphere vary widely in their molecular composition and chemical properties, so no single instrument can reasonably measure the entire range of ambient compounds. Over the past decade, a new generation of in situ, field-deployable mass spectrometers has dramatically improved our ability to detect, identify, and quantify these organic compounds, but no systematic approach has been developed to assess the extent to which currently available tools capture the entire space of chemical identity and properties that is expected in the atmosphere. Reduced-parameter frameworks that have been developed to describe atmospheric mixtures are exploited here to characterize the range of chemical properties accessed by a suite of instruments. Multiple chemical spaces (e.g. oxidation state of carbon vs. volatility, and oxygen number vs. carbon number) were populated with ions measured by several mass spectrometers, with gas- and particle-phase α-pinene oxidation products serving as the test mixture of organic compounds. Few gaps are observed in the coverage of the parameter spaces by the instruments employed in this work, though the full extent to which comprehensive measurement was achieved is difficult to assess due to uncertainty in the composition of the mixture. Overlaps between individual ions and regions in parameter space were identified, both between gas- and particle-phase measurements, and within each phase. These overlaps were conservatively found to account for little (<10%) of the measured mass. However, challenges in identifying overlaps and in accurately converting molecular formulas into chemical properties (such as volatility or reactivity) highlight a continued need to incorporate structural information into atmospheric measurements.

Low alanine aminotransferase levels and higher number of cardiovascular events in people with Type 2 diabetes: analysis of the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study.

AIMS: To determine whether alanine aminotransferase or gamma-glutamyltransferase levels, as markers of liver health and non-alcoholic fatty liver disease, might predict cardiovascular events in people with Type 2 diabetes. METHODS: Data from the Fenofibrate Intervention and Event Lowering in Diabetes study were analysed to examine the relationship between liver enzymes and incident cardiovascular events (non-fatal myocardial infarction, stroke, coronary and other cardiovascular death, coronary or carotid revascularization) over 5 years. RESULTS: Alanine aminotransferase measure had a linear inverse relationship with the first cardiovascular event occurring in participants during the study period. After adjustment, for every 1 sd higher baseline alanine aminotransferase measure (13.2 U/l), the risk of a cardiovascular event was 7% lower (95% CI 4-13; P = 0.02). Participants with alanine aminotransferase levels below and above the reference range 8-41 U/l for women and 9-59 U/l for men, had hazard ratios for a cardiovascular event of 1.86 (95% CI 1.12-3.09) and 0.65 (95% CI 0.49-0.87), respectively (P = 0.001). No relationship was found for gamma-glutamyltransferase. CONCLUSIONS: The data may indicate that in people with Type 2 diabetes, which is associated with higher alanine aminotransferase levels because of prevalent non-alcoholic fatty liver disease, a low alanine aminotransferase level is a marker of hepatic or systemic frailty rather than health.

A single prolonged milking interval of 24h compromises the well-being and health of dairy Holstein cows.

Cows are often shown at dairy shows with overfilled udders to achieve a better show placing. However, it is unclear to what degree "over-bagging" affects the health and well-being of show cows. The goal of this study was to assess the effect of a single prolonged milking interval (PMI) of 24h on the measurable signs of health and well-being in dairy cows in early and mid-lactation and to assess the effect of a nonsteroidal anti-inflammatory drug (NSAID) on well-being during a PMI. Fifteen Holstein cows were studied in early lactation (89.5±2.7d in milk) and were given an NSAID or physiological saline in a crossover design. Ten cows were studied again in mid-lactation (151.6±4.0d in milk). Data on clinical signs of cows' health, behavior, and well-being were collected at 1 or 2h intervals before and during a PMI of 24h. Data from the last 6h of a 12h milking interval were compared with the last 6h of the PMI. Compared with that of a cow in the last 6h of a 12-h milking interval, the behavior of cows in early lactation (saline group) changed during the last 6h of the PMI: we observed decreased eating time (22.4 vs. 16.2min/h), increased ruminating time (13.3 vs. 25.0min/h), and increased hind limb abduction while walking (score 41.7 vs. 62.6) and standing (31.2 vs. 38.9cm). Udder firmness was increased (2.9 vs. 4.5kg) during this period and more weight was placed on the hind limbs (46.4 vs. 47.0%). We also found pathological signs at the end of the PMI: all cows showed milk leaking, and 10 of 15 cows developed edema in the subcutaneous udder tissue. Somatic cell count was significantly increased from 12h to 72h after the PMI. Administration of an NSAID had no influence on measured variables, except that the occurrence of edema was not significantly increased during PMI in the flunixin group (10 of 15 and 6 of 15 cows for the saline and flunixin groups, respectively). In the cows in mid-lactation, different variables were not significantly changed in the PMI compared with baseline values (e.g., eating and ruminating time, occurrence of edema, and abduction). We conclude that the cows' health and well-being were compromised by a single PMI of 24h, because their behavior changed and pathological signs were recorded. Administration of an NSAID had a slight effect on cows' well-being during a PMI. The stage of lactation had more effect on the cows' health and well-being, because fewer variables were changed in mid-lactation.

A justification for less restrictive guidelines on the use of metformin in stable chronic renal failure.

AIM: The aim was to justify less restrictive use of metformin in stable chronic renal failure, because a literature review reveals metformin is associated with a significantly lower incidence of cardiovascular events and mortality compared with other hypoglycaemic agents, and metformin-associated lactic acidosis is rare and causation uncertain. Studies on intentional metformin overdose and metformin bioavailability, renal clearance and plasma metformin in renal impairment provide evidence in support of a less restrictive use of metformin. METHODS: In metformin overdose (n = 22), lactic acidosis was not inevitable with a plasma metformin > 40 mg/l (therapeutic level c. 1 mg/l): Severe lactic acidosis (pH ≤ 7.21, plasma lactate ≥ 11 mmol/l, n = 8) did not occur unless plasma metformin was > 40 mg/l. Plasma lactate was a more consistent predictor of pH than plasma metformin, with plasma lactate ≤ 4.7 being associated with a pH ≥ 7.34. A likely 'safe' plasma lactate is < 3.5 mmol/l and plasma metformin < 10 mg/l. RESULTS: Plasma metformin can be predicted from estimated glomerular filtration rate and metformin dose. Reported plasma metformin in renal failure was always less than predicted plasma metformin. Predicted plasma metformin (mg/l), with an estimated glomerular filtration rate of 30 ml/min and metformin 2000 mg/day was 6.8; an estimated glomerular filtration rate of 20 ml/min and metformin 1500 mg/day was 5.1; an estimated glomerular filtration rate of 10 ml/min and metformin 500 mg/day was 4.4. CONCLUSION: Metformin accumulates in renal failure and, although accumulation does not always lead to lactic acidosis, dose modification to achieve a predicted plasma metformin < 10 mg/l is suggested. As plasma metformin is not routinely available, plasma lactate should be useful in monitoring the use of metformin in renal failure.

Liver transplantation for the treatment of homozygous familial hypercholesterolaemia in an era of emerging lipid-lowering therapies.

Homozygous familial hypercholesterolaemia (FH) causes severe premature coronary artery disease because of very high levels of low density lipoprotein (LDL)-cholesterol. Standard lipid-lowering drugs and LDL-apheresis may not be sufficiently effective. Liver transplantation replaces defective LDL receptors and vastly improves the lipid profile, and we present the first report of an Australian adult to receive this treatment. Emerging drug treatments for FH may be alternatives to LDL-apheresis and transplantation, but long-term safety and efficacy data are lacking for all of these options.

Multiple functional polymorphisms in the G6PC2 gene contribute to the association with higher fasting plasma glucose levels.

AIMS/HYPOTHESIS: We previously identified the G6PC2 locus as a strong determinant of fasting plasma glucose (FPG) and showed that a common G6PC2 intronic single nucleotide polymorphism (SNP) (rs560887) and two common G6PC2 promoter SNPs (rs573225 and rs13431652) are highly associated with FPG. However, these promoter SNPs have complex effects on G6PC2 fusion gene expression, and our data suggested that only rs13431652 is a potentially causative SNP. Here we examine the effect of rs560887 on G6PC2 pre-mRNA splicing and the contribution of an additional common G6PC2 promoter SNP, rs2232316, to the association signal. METHODS: Minigene analyses were used to characterise the effect of rs560887 on G6PC2 pre-mRNA splicing. Fusion gene and gel retardation analyses characterised the effect of rs2232316 on G6PC2 promoter activity and transcription factor binding. The genetic association of rs2232316 with FPG variation was assessed using regression adjusted for age, sex and BMI in 4,220 Europeans with normal FPG. RESULTS: The rs560887-G allele was shown to enhance G6PC2 pre-mRNA splicing, whereas the rs2232316-A allele enhanced G6PC2 transcription by promoting Foxa2 binding. Genetic analyses provide evidence for association of the rs2232316-A allele with increased FPG (ß = 0.04 mmol/l; p = 4.3 × 10(-3)) as part of the same signal as rs560887, rs573225 and rs13431652. CONCLUSIONS/INTERPRETATION: As with rs13431652, the in situ functional data with rs560887 and rs2232316 are in accord with the putative function of G6PC2 in pancreatic islets, and suggest that all three are potentially causative SNPs that contribute to the association between G6PC2 and FPG.

A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene.

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E. Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14-15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18. The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

Management of acute poststroke blood pressure and detection of atrial fibrillation: a postal questionnaire of UK stroke physicians' current clinical practice.

The aim of this study was to determine the current clinical practice of UK stroke physicians with regard to the early management of blood pressure (BP) and arrhythmia detection following acute stroke. Postal service evaluation questionnaires were sent to the lead physicians for stroke in UK hospitals. Hospitals were identified by their inclusion in the 2008 Scottish Stroke Care Audit and the 2006 Royal College of Physicians Sentinel Stroke Audit. A total of 259 questionnaires were sent with a 33% response rate. Current practice regarding acute post-stroke BP management varied considerably. Approximately one-third of respondents lowered systolic BP within the first 72 hours of stroke, but the majority (65%) delayed intervening for at least seven days. Most would not intervene until systolic BP exceeded 180 mmHg. Of those who intervene, the most commonly quoted target systolic BP was 160 ± 5 mmHg. Post-stroke arrhythmia investigation was similarly varied; 12-lead electrocardiogram recording was frequent, with further investigation being more individualized. Of all respondents, 87% expressed interest in participating in future trials of complex interventions for stroke. Current practice of UK stroke physicians regarding acute BP intervention is diverse, reflecting conflicting evidence. There is interest in the stroke community for further research aiming to answer these important clinical questions.

Chasing Flies: The Use of Wingbeat Frequency as a Communication Cue in Calyptrate Flies (Diptera: Calyptratae).

The incidental sound produced by the oscillation of insect wings during flight provides an opportunity for species identification. Calyptrate flies include some of the fastest and most agile flying insects, capable of rapid changes in direction and the fast pursuit of conspecifics. This flight pattern makes the continuous and close recording of their wingbeat frequency difficult and limited to confined specimens. Advances in sound editor and analysis software, however, have made it possible to isolate low amplitude sounds using noise reduction and pitch detection algorithms. To explore differences in wingbeat frequency between genera and sex, 40 specimens of three-day old Sarcophaga crassipalpis, Lucilia sericata, Calliphora dubia, and Musca vetustissima were individually recorded in free flight in a temperature-controlled room. Results showed significant differences in wingbeat frequency between the four species and intersexual differences for each species. Discriminant analysis classifying the three carrion flies resulted in 77.5% classified correctly overall, with the correct classification of 82.5% of S. crassipalpis, 60% of C. dubia, and 90% of L. sericata, when both mean wingbeat frequency and sex were included. Intersexual differences were further demonstrated by male flies showing significantly higher variability than females in three of the species. These observed intergeneric and intersexual differences in wingbeat frequency start the discussion on the use of the metric as a communication signal by this taxon. The success of the methodology demonstrated differences at the genus level and encourages the recording of additional species and the use of wingbeat frequency as an identification tool for these flies.

Immunoregulatory cytokines are associated with protection from immunopathology following Mycobacterium avium subspecies paratuberculosis infection in red deer.

Although the causative agent of Johne's disease, Mycobacterium avium subsp. paratuberculosis, is well known, the etiology of disease and the immune responses generated in response to infection are still poorly understood. Knowledge of definitive markers of protective immunity, infection, and the establishment of chronic granulomatous Johne's disease is necessary to advance vaccine and diagnostic development. We sought to profile the immune responses occurring within jejunal lymph nodes of experimentally challenged red deer (Cervus elaphus). Quantitative PCR was utilized to measure a range of cytokines, signaling molecules, and transcription factors involved in Th1, Th2, Treg, and Th17 immune responses. Significant differences in gene expression were observed between control, minimally diseased, and severely diseased animals, with severely diseased animals showing elevated proinflammatory transcripts and reduced anti-inflammatory transcripts. We identified a proinflammatory cytokine milieu of gamma interferon, interleukin-1α (IL-1α), and IL-17, which may contribute to the immunopathology observed during clinical Johne's disease and suggest that Th2 and Treg immune responses may play an important role in controlling the development of immunopathology in infected animals.

Negative regulation of airway responsiveness that is dependent on gammadelta T cells and independent of alphabeta T cells.

The mechanisms regulating airway function are complex and still poorly understood. In diseases such as asthma, involvement of immune-dependent mechanisms has been suggested in causing changes in airway responsiveness to bronchoconstrictors. We now demonstrate that gammadelta T cells can regulate airway function in an alphabeta T cell-independent manner, identifying them as important cells in pulmonary homeostasis. This function of gammadelta T cells differs from previously described immune-dependent mechanisms and may reflect their interaction with innate systems of host defense.

Domestic Filth Flies in New Haven, Connecticut: A Case Study on the Effects of Urbanization and Climate Change by Comparing Fly Populations after 78 Years.

Changes in common and widespread insect populations such as the domestic filth fly in urban cities are useful and relevant bioindicators for overall changes in the insect biomass. The current study surveyed necrophagous flies by placing a weekly trap from June-September over a two-year period in the city of New Haven, Connecticut, to compare data on fly abundance and diversity with data collected 78 years earlier. Climate and land cover changes were also assessed in combination with the fly population for each period. The survey results suggest the domestic filth fly population is now less diverse with decreased species richness and changes in the relative abundance of species. In both surveys, 95-96% of the population was composed of only three species. The current survey data indicate the numerical dominance of Lucilia sericata has decreased, the abundance of several species, notably Lucilia coeruleiviridis, has increased, and Lucilia illustris is absent. Species that showed a significant interaction with temperature in the 1940s survey have now increased in abundance, with several of the trapped species continuing to show an interaction with temperature and rainfall. Analysis of the land cover and climate data characterizes the trap site as a region exposed to a prolonged period of industrialization and urbanization, with only 7% of the land cover remaining undeveloped and over 50% impervious, coupled with an increase in temperature and rainfall. This study serves as a model for changes in domestic filth fly populations and other insects in similarly highly urbanized established cities.

Forensically relevant blow flies (Diptera: Calliphoridae) of Central Connecticut, USA.

Connecticut and the Northeastern United States in general, lacks forensically relevant entomological survey data. To determine which forensically relevant calliphorid (blow flies, Diptera: Calliphoridae) and non-calliphorid species dominate Connecticut, 5 traps using pork bait were set out 7 times over a 2-year period to collect adult and immature specimens. Insects collected from human corpses in this region were also tabulated to collaborate the forensic relevance of trapped specimens. The survey identified a total of 8 species of blow flies of the Lucilia and Calliphora genera, 6 of which colonized the baits, and 5 of which have been found to colonize human corpses in the area. Non-calliphorid genera Sarcophaga and Muscina were also found to be colonizers of the baited traps but in relatively lower numbers. Trap sites differed significantly in the degree of urbanization which was determined by using GIS mapping to classify a 1 kilometer (km) radius around each site using land use and the percent of urban impervious surfaces. The 1 km radius revealed the highly fragmented nature of the immediate habitats of the trap sites and no habitat or seasonal preference was demonstrated by blow flies under these trap conditions. Temperature was the one variable which significantly affected the number of flies trapped and the colonizing species. All trapped species have been described previously as widespread and common in the United States and as synanthropic. Further research in this region should trap at the constantly changing extreme ends of the urban-rural gradient and in the colder temperatures of winter to explore the limits of the flexibility of these blow fly species.

Analysis of somatic mutations in kappa transgenes.

We have examined the nature and localization of somatic mutations in three kappa transgenes cloned from IgG-secreting hybridomas. All of the mutations identified were single base substitutions. Mutations were localized to the variable (V) region and its flanking sequences. In every case, the nuclear matrix association region, kappa enhancer, and C gene were spared. These data indicate that the rearranged kappa gene contains the necessary sequences for targeting of the mutation process, and suggest that the observed localization of mutations to the V region reflects the inherent specificity of this mutation process.