How does small group continuing medical education (CME) impact on practice for rural GPs and their patients, a mixed-methods study.

Studies which report outcomes of continuing medical education (CME) interventions for rural general practitioners (GPs) are limited. This mixed methods study recruited GPs from four CME small group learning (SGL) tutor groups based in different rural locations in the Republic of Ireland. A two-hour teaching module on deprescribing in older patients was devised and implemented. Assessment of educational outcomes was via questionnaires, prescribing audits and qualitative focus groups. All GPs (n = 43) in these CME-SGL groups agreed to participate, 27 of whom (63%) self-identified as being in rural practice. Rural GPs were more likely to be male (56%), in practice for longer (19 years), and attending CME for longer (13 years). The questionnaires indicated learning outcomes were achieved knowledge increased immediately after the education, and was maintained 6 months later. Twenty-four GPs completed audits involving 191 patients. Of these, 152 (79.6%) were de-prescribed medication. In the qualitative focus groups, GPs reported sharing experiences with their peers during CME-SGL helped them to improve patient care and ensured that clinical practice is more consistent across the group. For rural GPs, CME-SGL involving discussion of cases and the practical implementation of guidelines, associated with audit, can lead to changes in patient care.

Does locally delivered small group continuing medical education (CME) meet the learning needs of rural general practitioners?

The World Health Organisation policy of improving retention of rural health care practitioners recommended that continuing medical education (CME) programmes addressing their needs should be accessible and delivered where they live and work. This cross-sectional study involved a self-administered anonymous questionnaire completed by GPs attending CME small groups (CME-SGL) in Ireland. All GPs attending CME-SGL in a one-month period were invited to complete the questionnaire which examined demographic details, distance to travel to educational meetings/nearest regional hospital, barriers to accessing continuing education, whether CME-SGL met their educational needs, morale and professional isolation. A total of 1,686 responses were collected, of which 332 (19.6%) were from rural GPs. Of these, 289 (87%) reported that their educational needs were fully or mostly met by attending CME-SGL. Compared to urban doctors, rural GPs had further to travel to CME-SGL meetings, were further from the nearest regional hospital, and reported increased barriers to accessing continuing education. Rural GPs reported lower morale and greater levels of professional isolation. Despite considerable barriers to accessing continuing education, rural GPs reported that CME-SGL meets their educational needs. Future research should focus on the potential positive impact this may have on professional isolation and morale.

Irish Medical Students Understanding of the Intern Year.

Upon completion of medical school in Ireland, graduates must make the transition to becoming interns. The transition into the intern year may be described as challenging as graduates assume clinical responsibilities. Historically, a survey of interns in 1996 found that 91% felt unprepared for their role. However, recent surveys in 2012 have demonstrated that this is changing with preparedness rates reaching 52%. This can be partially explained by multiple initiatives at the local and national level. Our study aimed evaluate medical student understanding of the intern year and associated factors. An online, cross-sectional survey was sent out to all Irish medical students in 2013 and included questions regarding their understanding of the intern year. Two thousand, two hundred and forty-eight students responded, with 1,224 (55.4%) of students agreeing or strongly agreeing that they had a good understanding of what the intern year entails. This rose to 485 (73.7%) among senior medical students. Of junior medical students, 260 (42.8%) indicated they understood what the intern year, compared to 479 (48.7%) of intermediate medical students. Initiatives to continue improving preparedness for the intern year are essential in ensuring a smooth and less stressful transition into the medical workforce.

Ataxia telangiectasia: more variation at clinical and cellular levels.

Ataxia telangiectasia (A-T) is a rare recessively inherited disorder resulting in a progressive neurological decline. It is caused by biallelic mutation of the ATM gene that encodes a 370 kDa serine/threonine protein kinase responsible for phosphorylating many target proteins. ATM is activated by auto(trans)phosphorylation in response to DNA double strand breaks and leads to the activation of cell cycle checkpoints and either DNA repair or apoptosis as part of the cellular response to DNA damage. The allelic heterogeneity in A-T is striking. While the majority of mutations are truncating, leading to instability and loss of the ATM protein from the allele, a significant proportion of patients carry one of a small number of mutations that are either missense or leaky splice site mutations resulting in retention of some ATM with activity. The allelic heterogeneity in ATM, therefore, results in an equally striking clinical heterogeneity. There is also locus heterogeneity because mutation of the MRE11 gene can cause an obvious A-T like disorder both clinically and also at the cellular level and mutation of the RNF168 gene results in a much milder clinical phenotype, neurologically, with the major clinical feature being an immunological defect.

Medical students' views on selection tools for medical school--a mixed methods study.

It is important to ensure that the tools used in Medical School selection are acceptable to students and applicants. A questionnaire was administered to year 1 medical students in 2010 to determine the suitability of a variety of selection tools and the acceptability of HPAT-Ireland in particular. There were 291 respondents a 77% response rate representing approximately one third of all school leaver entrants that year. While the majority 285 (98%) were in favour of using school leaving examinations there was also support for the use of interviews 215 (74%) and other tools. Three quarters of Irish respondents 159 (76%) agreed that HPAT-Ireland is a fair test overall however section 3 (non-verbal reasoning) appeared less acceptable and relevant than other sections. A little over half had taken a preparatory HPAT-Ireland course 112 (54%). Medical school applicants appear to accept the use of non-traditional tools in the selection process.

Severe reaction to radiotherapy for breast cancer as the presenting feature of ataxia telangiectasia.

BACKGROUND: Severe early and late radiation reaction to radiotherapy is extremely rare in breast cancer patients. Such a reaction prompted an investigation into a 44-year-old mother (patient A-T213). METHODS: A neurological examination was performed and blood lymphocytes and skin fibroblasts were assessed for radiosensitivity chromosomally and by colony-forming assay. The ATM gene was sequenced and ATM mutations modelled by site-directed mutagenesis. The ATM kinase activity was also assessed. RESULTS: Patient A-T213 was normally ambulant with no ataxia and minimal other neurological features. T lymphocytes and skin fibroblasts were unusually radiosensitive, although less sensitive than in classical ataxia telangiectasia (A-T). A lymphoblastoid cell line and skin fibroblasts expressed ATM protein with some retained kinase activity. One missense ATM mutation c.8672G>A (p.Gly2891Asp) and a c.1A>G substitution were identified. In the modelling system, the p.Gly2891Asp mutant protein was expressed and shown to have residual ATM kinase activity. CONCLUSION: Patient A-T213 has a milder form of A-T with biallelic ATM mutations, which may have contributed to breast cancer development, and certainly caused the severe radiation reaction. Ataxia telangiectasia should be investigated as a potential cause of untoward severe early and late radiation reactions in breast cancer patients.

Early mechanisms of neutrophil activation and transmigration in acute lung injury.

Introduction:Neutrophil transmigration is multifactorial and primarily driven by selectins and ß2-integrins (CD11b/CD18), whose expression are dependent on the underlying stimulus. Ventilator-induced lung injury (VILI) results in a predominantly CD18-independent mechanism of neutrophil recruitment, while direct endotoxin-induced lung injury results from a CD18-dependent mechanism. We previously observed that lack of NADPH oxidases DUOX1 and DUOX2 resulted in reduced neutrophil influx in a VILI model of lung injury but had no influence on neutrophil influx after LPS exposure. Based on these observations, we hypothesized that DUOX1/DUOX2 are an important component of CD18-independent mechanisms of neutrophil recruitment in the lung. Methods:We exposed Duoxa -/- (KO) mice and Duoxa +/+ (WT) mice to either an intratracheal exposure of lipopolysaccharide (LPS/endotoxin)-or high tidal volume ventilation and compared expression of neutrophil markers between groups. WT mice (129S6/SvEvTac) were obtained from Taconic Biosciences (One Discovery Drive Suite 304; Rensselaer, NY 1244) and were allowed to acclimatize for one week prior to study enrollment. KO mice were generated as previously described [Grasberger 2012] and bred in-house on a 129S6 background. We provided positive-pressure ventilation at a tidal volume of 10 ml/kg with 2 cmH20 positive end-expiratory pressure (PEEP). Mice were assigned to groups consisting of KO (n = 5) and WT (n = 5) in each group and divided into non-ventilated, positive-pressure ventilation, or LPS IT exposure groups. Positive-pressure ventilation was instituted for 4-h using a FlexiVent (Flexiware 8.1, Scireq, Montreal, QC, Canada). Lipopolysaccharide (Salmonella enterica serotype tryphimurium L6143, Millipore Sigma) was administered via an intratracheal (IT) route at a dose of 0.1 mg/kg. Mice were humanely euthanized at 4-h post-injection consistent with the UC Davis IAUCAC-approved protocol. Results:As previously observed, neutrophilic influx into the airways was significantly impaired in the Duoxa -/- (KO) mice after VILI, but not after LPS exposure. LPS-induced lung injury resulted in upregulation of CD11b+ neutrophils and shedding of CD62L and CD162 regardless of DUOX expression, whereas VILI resulted in upregulation of CD49+ neutrophils in the Duoxa +/+ (WT) mice but not the Duoxa -/- (KO) mice. Conclusion:Our data suggest DUOX is required for CD18-independent mechanisms of neutrophil recruitment in the lung induced by acute lung injury, but not for canonical CD18depedent mechanisms after LPS exposure.

Lymphoid tumours and breast cancer in ataxia telangiectasia; substantial protective effect of residual ATM kinase activity against childhood tumours.

BACKGROUND: Immunodeficiency in ataxia telangiectasia (A-T) is less severe in patients expressing some mutant or normal ATM kinase activity. We, therefore, determined whether expression of residual ATM kinase activity also protected against tumour development in A-T. METHODS: From a total of 296 consecutive genetically confirmed A-T patients from the British Isles and the Netherlands, we identified 66 patients who developed a malignant tumour; 47 lymphoid tumours and 19 non-lymphoid tumours were diagnosed. We determined their ATM mutations, and whether cells from these patients expressed any ATM with residual ATM kinase activity. RESULTS: In childhood, total absence of ATM kinase activity was associated, almost exclusively, with development of lymphoid tumours. There was an overwhelming preponderance of tumours in patients <16 years without kinase activity compared with those with some residual activity, consistent with a substantial protective effect of residual ATM kinase activity against tumour development in childhood. In addition, the presence of eight breast cancers in A-T patients, a 30-fold increased risk, establishes breast cancer as part of the A-T phenotype. CONCLUSION: Overall, a spectrum of tumour types is associated with A-T, consistent with involvement of ATM in different mechanisms of tumour formation. Tumour type was influenced by ATM allelic heterogeneity, residual ATM kinase activity and age.

Phage anti-immunocomplex assay for clomazone: two-site recognition increasing assay specificity and facilitating adaptation into an on-site format.

The impact of the use of herbicides in agriculture can be minimized by compliance with good management practices that reduce the amount used and their release into the environment. Simple tests that provide real time on-site information about these chemicals are a major aid for these programs. In this work, we show that phage anti-immunocomplex assay (PHAIA), a method that uses phage-borne peptides to detect the formation of antibody-analyte immunocomplexes, is an advantageous technology to produce such field tests. A monoclonal antibody to the herbicide clomazone was raised and used in the development of conventional competitive and noncompetitive PHAIA immunoassays. The sensitivity attained with the PHAIA format was over 10 times higher than that of the competitive format. The cross-reactivity of the two methods was also compared using structurally related compounds, and we observed that the two-site binding of PHAIA "double-checks" the recognition of the analyte, thereby increasing the assay specificity. The positive readout of the noncompetitive PHAIA method allowed adaptation of the assay into a rapid and simple format where as little as 0.4 ng/mL clomazone (more than 10-fold lower than the proposed standard) in water samples from a rice field could be easily detected by simple visual inspection.

High-throughput method for ranking the affinity of peptide ligands selected from phage display libraries.

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major "bottleneck" of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred "en masse" from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide-BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide-BAP protein can find direct application as a tracer reagent.

A method to establish the relationship between chronological age and stage of union from radiographic assessment of epiphyseal fusion at the knee: an Irish population study.

Characteristic changes during epiphyseal union provide a skeletal age, which when compared with age-based standards provides an estimation of chronological age. Currently there are no data on epiphyseal union for the purposes of age estimation specific to an Irish population. This cross-sectional study aims to investigate the relationship between stage of epiphyseal union at the knee joint and chronological age in a modern Irish population. A novel radiographic method that sub-divides the continuum of development into five specific stages of union is presented. Anteroposterior and lateral knee radiographs of 148 males and 86 females, aged 9-19 years, were examined. Fusion was scored as Stage 0, non-union; Stage 1, beginning union; Stage 2, active union; Stage 3, recent union; or Stage 4, complete union. Stage of epiphyseal union is correlated with chronological age in both males and females. Mean age gradually increases with each stage of union and also varies between male and female subjects. A statistically significant difference in mean age was recorded between stages when compared to the previous stage, for the three epiphyses. Irish children are comparable to those from previously published studies with epiphyseal union in females occurring earlier than males. A significant difference was noted between the mean age of union for males and females for each of Stages 1 and 2 for the femur and Stages 0, 1, 2 and 3 for the tibia and the fibula. The results also suggest that the stages of union occur at earlier ages in this Irish population. Implementation of standardized methodology is necessary to investigate if this is due to a secular or population variation in maturation or to a methodology which clearly identifies five stages of union.

The introduction of mandatory CPD for newly state registered diagnostic radiographers: An Irish perspective.

INTRODUCTION: Irish diagnostic radiography has undergone significant change with the dual introduction of state registration and mandatory Continuing Professional Development (CPD) in October 2015. We aimed to investigate motivators and barriers around CPD participation, mechanisms of CPD delivery and confidence of radiographers in using e-learning. METHODS: A questionnaire distributed nationally during this period captured Radiographer opinion through the use of closed and open questions. The questionnaire was distributed in hard copy and online formats depending on site preference. RESULTS: 71% of centres participated, rendering 453 responses in total from a possible 1222 respondents employed in those sites at the time of the survey. A varied range of ages and post qualification experience were represented. Respondents indicated use of several CPD options with the majority considering CPD to be important, very important or critical. Social media as a mechanism of CPD delivery was considered acceptable by 48%, while online learning elicited responses ranging from not confident to absolutely confident. Top motivators for CPD activity included interest, developing new knowledge and competency. Principal barriers included funding, time allocation and location issues. CONCLUSION: This study identified Radiographer desire to undertake CPD and the need for developing online CPD offerings. An overall positive perception towards CPD was noted however barriers were identified which require specific redress strategies.

Mouse models of asthma: can they give us mechanistic insights into the role of nitric oxide?

New clinical practice guidelines for patients with asthma include the recommendation to monitor exhaled breath nitric oxide (NO) levels. NO concentrations in exhaled breath are increased in asthmatics and increased NO levels correlate with worsening airway inflammation and asthma symptoms. The multiple roles of NO in the lung have not been delineated clearly. Clinical trials are being performed presently that test the apparently conflicting hypotheses that either donors or inhibitors of NO in the lung are effective strategies for treating asthma. These strategies evolved, in part, from results of pre-clinical studies performed in mice and other animal models. This review evaluates the existing literature with regard to mouse models of asthma and explores the often conflicting data on the role of NO, the nitric oxide synthase (NOS) enzymes, and the arginase enzymes in allergic airway inflammation. While we will emphasize the ovalbumin exposure mouse model, we will also examine other models. Where inconsistencies are identified among the studies, we attempt to determine whether such inconsistencies arise from methodological differences or alternative mechanisms. Ultimately, we address whether the allergen-exposed mouse is a suitable model for identifying promising new drugs for the treatment of human asthma. While a consensus is building that NO is beneficial or protective in subsets of asthmatics, results from studies using mouse models to investigate the individual roles of NO and the NOS enzymes in airway inflammation are often contradictory. Further research efforts with this model will allow us to distinguish which asthma patients may benefit best from NO donors and which may benefit from NO inhibitors.

Workshop summary: phosgene-induced pulmonary toxicity revisited: appraisal of early and late markers of pulmonary injury from animal models with emphasis on human significance.

A workshop was held February 14, 2007, in Arlington, VA, under the auspices of the Phosgene Panel of the American Chemistry Council. The objective of this workshop was to convene inhalation toxicologists and medical experts from academia, industry and regulatory authorities to critically discuss past and recent inhalation studies of phosgene in controlled animal models. This included presentations addressing the benefits and limitations of rodent (mice, rats) and nonrodent (dogs) species to study concentration x time (C x t) relationships of acute and chronic types of pulmonary changes. Toxicological endpoints focused on the primary pulmonary effects associated with the acute inhalation exposure to phosgene gas and responses secondary to injury. A consensus was reached that the phosgene-induced increased pulmonary extravasation of fluid and protein can suitably be probed by bronchoalveolar lavage (BAL) techniques. BAL fluid analyses rank among the most sensitive methods to detect phosgene-induced noncardiogenic, pulmonary high-permeability edema following acute inhalation exposure. Maximum protein concentrations in BAL fluid occurred within 1 day after exposure, typically followed by a latency period up to about 15 h, which is reciprocal to the C x t exposure relationship. The C x t relationship was constant over a wide range of concentrations and single exposure durations. Following intermittent, repeated exposures of fixed duration, increased tolerance to recurrent exposures occurred. For such exposure regimens, chronic effects appear to be clearly dependent on the concentration rather than the cumulative concentration x time relationship. The threshold C x t product based on an increased BAL fluid protein following single exposure was essentially identical to the respective C x t product following subchronic exposure of rats based on increased pulmonary collagen and influx of inflammatory cells. Thus, the chronic outcome appears to be contingent upon the acute pulmonary threshold dose. Exposure concentrations high enough to elicit an increased acute extravasation of plasma constituents into the alveolus may also be associated with surfactant dysfunction, intra-alveolar accumulation of fibrin and collagen, and increased recruitment and activation of inflammatory cells. Although the exact mechanisms of toxicity have not yet been completely elucidated, consensus was reached that the acute pulmonary toxicity of phosgene gas is consistent with a simple, irritant mode of action at the site of its initial deposition/retention. The acute concentration x time mortality relationship of phosgene gas in rats is extremely steep, which is typical for a local, directly acting pulmonary irritant gas. Due to the high lipophilicity of phosgene gas, it efficiently penetrates the lower respiratory tract. Indeed, more recent published evidence from animals or humans has not revealed appreciable irritant responses in central and upper airways, unless exposure was to almost lethal concentrations. The comparison of acute inhalation studies in rats and dogs with focus on changes in BAL fluid constituents demonstrates that dogs are approximately three to four times less susceptible to phosgene than rats under methodologically similar conditions. There are data to suggest that the dog may be useful particularly for the study of mechanisms associated with the acute extravasation of plasma constituents because of its size and general morphology and physiology of the lung as well as its oronasal breathing patterns. However, the study of the long-term sequelae of acute effects is experimentally markedly more demanding in dogs as compared to rats, precluding the dog model to be applied on a routine base. The striking similarity of threshold concentrations from single exposure (increased protein in BAL fluid) and repeated-exposure 3-mo inhalation studies (increased pulmonary collagen deposition) in rats supports the notion that chronic changes depend on acute threshold mechanisms.

Modification by ozone of lung tumor development in mice.

Mice, either strain A/J or Swiss Webster, were exposed for 18 weeks either to filtered air or to 0.4 or 0.8 ppm ozone for 8 hours daily. Subgroups in each test group received a single ip injection of 1,000 mg urethan/kg or 0.9% sodium chloride vehicle 1 day prior to initiation of the exposure regimen. Tumor incidence in Swiss Webster mice was 0-3% in groups not receiving urethan and was 61-74% in groups receiving urethan. In A/J mice, the corresponding values were 9-38% and 100%, respectively. Exposure to ozone caused a decrease in the number of tumors per lung in urethan-treated mice of both strains, in a dose-dependent manner. There seemed to be a specific decrease in tumors derived from alveolar type II cells in the A/J mice given urethan plus ozone. Most interesting, perhaps, was a significant increase in the number of tumors per lung in A/J mice exposed to 0.8 ppm ozone without urethan, confirming a previous report by others. The corresponding ozone effect on lung tumor development was not observed in Swiss Webster mice.

The relevance of basic sciences in undergraduate medical education.

BACKGROUND: Evolving and changing undergraduate medical curricula raise concerns that there will no longer be a place for basic sciences. National and international trends show that 5-year programmes with a pre-requisite for school chemistry are growing more prevalent. National reports in Ireland show a decline in the availability of school chemistry and physics. AIM: This observational cohort study considers if the basic sciences of physics, chemistry and biology should be a prerequisite to entering medical school, be part of the core medical curriculum or if they have a place in the practice of medicine. METHODS: Comparisons of means, correlation and linear regression analysis assessed the degree of association between predictors (school and university basic sciences) and outcomes (year and degree GPA) for entrants to a 6-year Irish medical programme between 2006 and 2009 (n = 352). RESULTS: We found no statistically significant difference in medical programme performance between students with/without prior basic science knowledge. The Irish school exit exam and its components were mainly weak predictors of performance (-0.043 ≥ r ≤ 0.396). Success in year one of medicine, which includes a basic science curriculum, was indicative of later success (0.194 ≥ r (2) ≤ 0.534). CONCLUSIONS: University basic sciences were found to be more predictive than school sciences in undergraduate medical performance in our institution. The increasing emphasis of basic sciences in medical practice and the declining availability of school sciences should mandate medical schools in Ireland to consider how removing basic sciences from the curriculum might impact on future applicants.

Rat lysyl hydroxylase: molecular cloning, mRNA distribution and expression in a baculovirus system.

A cDNA library from rat lung was screened with a chicken lysyl hydroxylase cDNA, and several overlapping rat lysyl hydroxylase cDNAs were isolated. The complete cDNA was 91 and 77% identical, respectively, to the human and chicken lysyl hydroxylase cDNAs at the protein level. By Northern blot, the rat lysyl hydroxylase cDNA recognized a single 3.2 kb mRNA that was present in a wide variety of rat tissues. In order to further confirm the identity of this cDNA, the cDNA was expressed in insect cells via a baculovirus vector. These cells produced an 85 kDa protein with lysyl hydroxylase activity. The recombinant lysyl hydroxylase had a specific activity and Km values for its substrates that were similar to those of the enzyme isolated from chick embryos. The fact that this single lysyl hydroxylase cDNA encodes a protein sufficient for lysyl hydroxylase activity is consistent with previous biochemical findings that lysyl hydroxylase only requires a single type of subunit for its activity.

Biosynthesis of collagen crosslinks. II. In vivo labelling and stability of lung collagen in rats.

Rat lung collagen was labelled in vivo by a single intraperitoneal injection of [3H]lysine at several key timepoints in lung development: days 11 (alveolar proliferation), 26 (start of equilibrated growth), 42 (end of equilibrated growth), and 100 (adult lung structure present). The rates of deposition of labelled hydroxylysine and the difunctional, Schiff base-derived crosslinks hydroxylysinonorleucine (HLNL) and dihydroxylysinonorleucine (DHLNL) were quantified. We also measured total lung content of the trifunctional, mature crosslink hydroxypyridinium (OHP) in these same animals. While the relative rates of accumulation of labelled collagen [3H]hydroxylysine differed by a factor of about 6 at the different times of injection of labelled precursor, quantitative and qualitative patterns of collagen crosslinking were very similar at all of the lung developmental stages studied. Furthermore, there was little or no breakdown of the lung collagen pool as defined by the presence of labelled crosslinks; changes in lung DHLNL content could be completely accounted for by its maturation to OHP, regardless of the age of the rats when injected with the radioactive precursor. We conclude that mature, crosslinked collagen in the lungs of rats, which is obligatorily an extracellular pool, is not being degraded at a measurable rate. Therefore, studies of others that have shown apparent high rates of breakdown of newly synthesized collagen in lungs of whole animals using different methods are probably not reflective of the metabolic fate of total lung collagen, and may indicate that degradation of normal lung collagen occurs predominantly or exclusively intracellularly.

Analysis of age-associated changes in collagen crosslinking in the skin and lung in monkeys and rats.

The present study was designed to address a specific question: can we define collagen aging in vivo in terms of alterations in collagen crosslinking? In order to assess the complete spectrum of change throughout life, tissues from rats, monkeys and (where available) humans were examined at ages ranging from fetal to old. Skin and lung were selected in order to include all of the crosslinks derived from lysyl oxidase-generated aldehydes that have been identified thus far, both reducible and nonreducible. Crosslinks analyzed included hydroxylysinonorleucine, dihydroxylysinorleucine, histidinohydroxymerodesmosine, hydroxypyridinium, lysyl pyridinium, and a deoxy analogue of hydroxypyridinium found in skin that differs structurally from lysyl pyridinium. Tissues from both a short-lived species (rats) and a long-lived species (monkeys) were analyzed to test further the hypothesis that changes in crosslinking are linked predominantly to biological age of the animal, rather than temporal aging. We found that biological aging seems to regulate certain predictable changes during the first part of the lifespan: the disappearance postnatally of dihydroxylysinonorleucine in skin, the rapid decrease in difunctional crosslink content in lung and skin during early growth and development, and the gradual rise in hydroxypyridinium and lysyl pyridinium in lung tissue. Changes in crosslinking were far less predictable during the second half of the lifespan. Although hydroxypridinium content continued to rise or reached a plateau in rat and monkey lungs, respectively, it showed a decrease in human lungs. The analogous trifunctional crosslink in skin, the so-called 'pyridinoline analogue', decreased dramatically in both rats and monkeys in later life. Our data suggest that caution must be taken in drawing inferences about human connective tissue aging from experiments performed in short-lived species such as rodents. Furthermore, the finding that there may be fewer total lysyl oxidase-derived crosslinks per collagen molecule in very old animals as compared with young animals suggests that we may need to expand our concepts of collagen crosslinking.

Lung collagen cross-links in rats with experimentally induced pulmonary fibrosis.

Rats were intratracheally instilled with bleomycin or with silica (quartz) dust to induce lung fibrosis. Several weeks later, purified collagen chains (or collagen digests) were isolated from the lungs of these animals and from age-matched controls instilled intratracheally with saline solution, and the ratios of hydroxylysine to lysine and of the dysfunctional cross-links DHLNL to HLNL were quantified. Collagen from fibrotic lungs had significantly higher ratios of DHLNL:HLNL than did control lungs, 15.5 +/- 4.8 and 17.1 +/- 4.8 vs. 2.3 +/- 0.5 for the silica-instilled and the bleomycin-instilled animals, respectively. The hydroxylysine:lysine ratio was significantly increased for the alpha 1(I) chain, to a value 170% of that of lung collagen from control animals, and for several of its constituent CNBr peptides. Lung tissue was exhaustively digested with collagenase and specific cross-linked peptides were isolated and characterized. The cross-linked alpha 1(I) x alpha 1(I) peptide linked by the residues 87 x 16C, with a ratio of DHLNL:HLNL of 17:1, demonstrated that the increased hydroxylation of the dysfunctional cross-links in fibrotic lung collagen could be accounted for in part by increased hydroxylation of the lysine residue at position 16C of the C-terminal telopeptide of the collagen alpha 1(I) chain. It proved impossible to locate the corresponding N-terminal cross-linked fragment from alpha 1(I) x alpha 1(I) chains, 9N x 930, possibly due to further reactions of this material to form the material referred to as poly(CB6). Isolated poly (CB6) accounted for more than half of the total alpha 1(I)CB6 peptide expected in lung collagen, and had a hydroxylysine:lysine content 2.8 times greater in bleomycin-treated animals than in their age-matched controls. Evidence was also found for a cross-linked alpha 1(III) x alpha 1(I) peptide linking residue 87 from the alpha 1(III) chain with residue 16C from the alpha 1(I) chain; it also had an increased ratio of DHLNL:HLNL. We conclude that the increased hydroxylation of lysine observed in two different animal models of lung fibrosis occurs preferentially at the N- and C-terminal nonhelical extension peptides of the alpha 1(I) collagen chains, and that this apparent specificity of overhydroxylation of fibrotic collagen may have important structural and pathological consequences.