Selective Targeting of Protein Kinase C (PKC)-θ Nuclear Translocation Reduces Mesenchymal Gene Signatures and Reinvigorates Dysfunctional CD8+ T Cells in Immunotherapy-Resistant and Metastatic Cancers.

Protein kinase C (PKC)-θ is a serine/threonine kinase with both cytoplasmic and nuclear functions. Nuclear chromatin-associated PKC-θ (nPKC-θ) is increasingly recognized to be pathogenic in cancer, whereas its cytoplasmic signaling is restricted to normal T-cell function. Here we show that nPKC-θ is enriched in circulating tumor cells (CTCs) in patients with triple-negative breast cancer (TNBC) brain metastases and immunotherapy-resistant metastatic melanoma and is associated with poor survival in immunotherapy-resistant disease. To target nPKC-θ, we designed a novel PKC-θ peptide inhibitor (nPKC-θi2) that selectively inhibits nPKC-θ nuclear translocation but not PKC-θ signaling in healthy T cells. Targeting nPKC-θ reduced mesenchymal cancer stem cell signatures in immunotherapy-resistant CTCs and TNBC xenografts. PKC-θ was also enriched in the nuclei of CD8+ T cells isolated from stage IV immunotherapy-resistant metastatic cancer patients. We show for the first time that nPKC-θ complexes with ZEB1, a key repressive transcription factor in epithelial-to-mesenchymal transition (EMT), in immunotherapy-resistant dysfunctional PD1+/CD8+ T cells. nPKC-θi2 inhibited the ZEB1/PKC-θ repressive complex to induce cytokine production in CD8+ T cells isolated from patients with immunotherapy-resistant disease. These data establish for the first time that nPKC-θ mediates immunotherapy resistance via its activity in CTCs and dysfunctional CD8+ T cells. Disrupting nPKC-θ but retaining its cytoplasmic function may offer a means to target metastases in combination with chemotherapy or immunotherapy.

Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans.

The diversity of circulating human B cells is unknown. We use single-cell RNA sequencing (RNA-seq) to examine the diversity of both antigen-specific and total B cells in healthy subjects and malaria-exposed individuals. This reveals two B cell lineages: a classical lineage of activated and resting memory B cells and an alternative lineage, which includes previously described atypical B cells. Although atypical B cells have previously been associated with disease states, the alternative lineage is common in healthy controls, as well as malaria-exposed individuals. We further track Plasmodium-specific B cells after malaria vaccination in naive volunteers. We find that alternative lineage cells are primed after the initial immunization and respond to booster doses. However, alternative lineage cells develop an atypical phenotype with repeated boosts. The data highlight that atypical cells are part of a wider alternative lineage of B cells that are a normal component of healthy immune responses.

Targeting Nuclear LSD1 to Reprogram Cancer Cells and Reinvigorate Exhausted T Cells via a Novel LSD1-EOMES Switch.

Lysine specific demethylase 1 (LSD1) is a key epigenetic eraser enzyme implicated in cancer metastases and recurrence. Nuclear LSD1 phosphorylated at serine 111 (nLSD1p) has been shown to be critical for the development of breast cancer stem cells. Here we show that circulating tumor cells isolated from immunotherapy-resistant metastatic melanoma patients express higher levels of nLSD1p compared to responders, which is associated with co-expression of stem-like, mesenchymal genes. Targeting nLSD1p with selective nLSD1 inhibitors better inhibits the stem-like mesenchymal signature than traditional FAD-specific LSD1 catalytic inhibitors such as GSK2879552. We also demonstrate that nLSD1p is enriched in PD-1+CD8+ T cells from resistant melanoma patients and 4T1 immunotherapy-resistant mice. Targeting the LSD1p nuclear axis induces IFN-γ/TNF-α-expressing CD8+ T cell infiltration into the tumors of 4T1 immunotherapy-resistant mice, which is further augmented by combined immunotherapy. Underpinning these observations, nLSD1p is regulated by the key T cell exhaustion transcription factor EOMES in dysfunctional CD8+ T cells. EOMES co-exists with nLSD1p in PD-1+CD8+ T cells in resistant patients, and nLSD1p regulates EOMES nuclear dynamics via demethylation/acetylation switching of critical EOMES residues. Using novel antibodies to target these post-translational modifications, we show that EOMES demethylation/acetylation is reciprocally expressed in resistant and responder patients. Overall, we show for the first time that dual inhibition of metastatic cancer cells and re-invigoration of the immune system requires LSD1 inhibitors that target the nLSD1p axis.

Cervical spine kinematics during machine-based and live scrummaging.

The aim of this study was to compare cervical spine kinematics in rugby union front row players during machine-based and "live" scrummaging. Cervical spine kinematics was measured via electromagnetic tracking of sensors attached to the head and thorax. Joint angles were extracted from each trial at two time points ("bind" prior to engagement and instant of impact) for comparison between scrummaging conditions. The effect of scrummaging condition on kinematics was evaluated using a mixed effects model and estimations were based on a Bayesian framework. With differences ranging from 38° to 50°, the results show that the cervical spine is consistently more flexed when scrummaging against opponents than against a scrum machine. In contrast, there are little differences in the excursion of lateral-flexion (range 5-8°) and axial rotation (7°) between the two conditions. The findings from this study provide clear information on motion patterns in different scrum formations, and suggest that the current design of scrum machines may not promote the same pattern of movement that occurs in live scrums. The results highlight that findings from previous studies that have investigated kinematics during machine-based scrummaging may not be generalisable to a competitive scrummaging context.

Physical characteristics associated with neck pain and injury in rugby union players.

BACKGROUND: Neck pain and injury are common in rugby union. Physical characteristics predisposing players to neck injury are largely unknown. This study aimed to determine physical characteristics associated with neck pain and injury in rugby union players. METHODS: Semi-professional rugby union players (N.=142) underwent pre-season measurements including cervical active range of motion (AROM), strength, sensorimotor proprioception (joint position error), and anthropometry. A structured interview established previous neck injury history, current symptoms, playing position, competition level, age, and years playing rugby. Team physiotherapists and player telephone interviews identified players sustaining a neck injury during the competitive season (defined as any reported neck pain or neck injury). T-tests or Mann-Whitney U tests determined differences between neck injured and non-injured players. Logistic regression determined factors associated with neck injury history and incidence. RESULTS: Sixty-five (46%) players reported a previous neck injury; 11 (8%) sustained a neck injury during the competitive season. Player age (OR 1.14, 95% CI 1.03-1.25, P=0.009) was associated with neck injury history. Pre-season lateral flexion AROM was less in players sustaining a neck injury or reporting neck pain during the season (median left 23.6°, IQR 21.8-26.2°; right 27.9°, 23.6-32.5°) than in other players (left 34.8°, 28.8-41.0°, P<0.01; right 39.1°, 28.9-48.1°, P=0.03). Lateral flexion AROM was associated with increased risk of neck pain or injury (OR 0.82, 95%CI 0.71-0.94, P=0.005). CONCLUSIONS: Decreased cervical lateral flexion AROM may contribute to neck injury risk in rugby union players. However, few physical characteristics predicted neck injury incidence, suggesting additional factors should be explored to determine injury risk.

Nuclear PKC-θ facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation.

Memory T cells are characterized by their rapid transcriptional programs upon re-stimulation. This transcriptional memory response is facilitated by permissive chromatin, but exactly how the permissive epigenetic landscape in memory T cells integrates incoming stimulatory signals remains poorly understood. By genome-wide ChIP-sequencing ex vivo human CD4(+) T cells, here, we show that the signaling enzyme, protein kinase C theta (PKC-θ) directly relays stimulatory signals to chromatin by binding to transcriptional-memory-responsive genes to induce transcriptional activation. Flanked by permissive histone modifications, these PKC-enriched regions are significantly enriched with NF-κB motifs in ex vivo bulk and vaccinia-responsive human memory CD4(+) T cells. Within the nucleus, PKC-θ catalytic activity maintains the Ser536 phosphorylation on the p65 subunit of NF-κB (also known as RelA) and can directly influence chromatin accessibility at transcriptional memory genes by regulating H2B deposition through Ser32 phosphorylation. Furthermore, using a cytoplasm-restricted PKC-θ mutant, we highlight that chromatin-anchored PKC-θ integrates activating signals at the chromatin template to elicit transcriptional memory responses in human memory T cells.

The B subunit of Escherichia coli heat-labile toxin alters the development and antigen-presenting capacity of dendritic cells.

Escherichia coli's heat-labile enterotoxin (Etx) and its non-toxic B subunit (EtxB) have been characterized as adjuvants capable of enhancing T cell responses to co-administered antigen. Here, we investigate the direct effect of intravenously administered EtxB on the size of the dendritic and myeloid cell populations in spleen. EtxB treatment appears to enhance the development and turnover of dendritic and myeloid cells from precursors within the spleen. EtxB treatment also gives a dendritic cell (DC) population with higher viability and lower activation status based on the reduced expression of MHC-II, CD80 and CD86. In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide. In in vitro bone marrow cultures, EtxB treatment was also found to enhance the development of DC from precursors dependent on Flt3L. In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c(+) DC. In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells.

Novel and rare functional genomic variants in multiple autoimmune syndrome and Sjögren's syndrome.

BACKGROUND: Multiple autoimmune syndrome (MAS), an extreme phenotype of autoimmune disorders, is a very well suited trait to tackle genomic variants of these conditions. Whole exome sequencing (WES) is a widely used strategy for detection of protein coding and splicing variants associated with inherited diseases. METHODS: The DNA of eight patients affected by MAS [all of whom presenting with Sjögren's syndrome (SS)], four patients affected by SS alone and 38 unaffected individuals, were subject to WES. Filters to identify novel and rare functional (pathogenic-deleterious) homozygous and/or compound heterozygous variants in these patients and controls were applied. Bioinformatics tools such as the Human gene connectome as well as pathway and network analysis were applied to test overrepresentation of genes harbouring these variants in critical pathways and networks involved in autoimmunity. RESULTS: Eleven novel and rare functional variants were identified in cases but not in controls, harboured in: MACF1, KIAA0754, DUSP12, ICA1, CELA1, LRP1/STAT6, GRIN3B, ANKLE1, TMEM161A, and FKRP. These were subsequently subject to network analysis and their functional relatedness to genes already associated with autoimmunity was evaluated. Notably, the LRP1/STAT6 novel mutation was homozygous in one MAS affected patient and heterozygous in another. LRP1/STAT6 disclosed the strongest plausibility for autoimmunity. LRP1/STAT6 are involved in extracellular and intracellular anti-inflammatory pathways that play key roles in maintaining the homeostasis of the immune system. Further; networks, pathways, and interaction analyses showed that LRP1 is functionally related to the HLA-B and IL10 genes and it has a substantial impact within immunological pathways and/or reaction to bacterial and other foreign proteins (phagocytosis, regulation of phospholipase A2 activity, negative regulation of apoptosis and response to lipopolysaccharides). Further, ICA1 and STAT6 were also closely related to AIRE and IRF5, two very well known autoimmunity genes. CONCLUSIONS: Novel and rare exonic mutations that may account for autoimmunity were identified. Among those, the LRP1/STAT6 novel mutation has the strongest case for being categorised as potentially causative of MAS given the presence of intriguing patterns of functional interaction with other major genes shaping autoimmunity.

Chromatinized protein kinase C-θ directly regulates inducible genes in epithelial to mesenchymal transition and breast cancer stem cells.

Epithelial to mesenchymal transition (EMT) is activated during cancer invasion and metastasis, enriches for cancer stem cells (CSCs), and contributes to therapeutic resistance and disease recurrence. Signal transduction kinases play a pivotal role as chromatin-anchored proteins in eukaryotes. Here we report for the first time that protein kinase C-theta (PKC-θ) promotes EMT by acting as a critical chromatin-anchored switch for inducible genes via transforming growth factor ß (TGF-ß) and the key inflammatory regulatory protein NF-κB. Chromatinized PKC-θ exists as an active transcription complex and is required to establish a permissive chromatin state at signature EMT genes. Genome-wide analysis identifies a unique cohort of inducible PKC-θ-sensitive genes that are directly tethered to PKC-θ in the mesenchymal state. Collectively, we show that cross talk between signaling kinases and chromatin is critical for eliciting inducible transcriptional programs that drive mesenchymal differentiation and CSC formation, providing novel mechanisms to target using epigenetic therapy in breast cancer.

Regional bone geometry of the tibia in triathletes and stress reactions--an observational study.

OBJECTIVES: The association between tibial morphology and tibial stress fractures or tibial stress syndrome was examined in triathletes with an unusually high incidence of these injuries. DESIGN: A cross-sectional study design examined associations between tibial geometry from MRI images and training and injury data between male and female triathletes and between stress fracture (SF) and non-stress fracture (NSF) groups. METHODS: Fifteen athletes (7 females, 8 males) aged 17-23 years who were currently able to train and race were recruited from the New Zealand Triathlete Elite Development Squad. Geometric measurements were taken at 5 zones along the tibia using MRI and compared between symptomatic and asymptomatic tibiae subjects. RESULTS: SF tibiae displayed either oedema within the cancellous bone and/or stress fracture on MRI. When collapsed across levels, symptomatic tibiae had thicker medial cortices (F1,140=9.285, p=0.003), thicker lateral cortices (F1,140=10.129, p=0.002) and thinner anterior cortices (F1,140=14.517, p=0.000) than NSF tibiae. Only medial cortex thickness in SF tibia was significantly different (F4,140=3.358, p=0.012) at different levels. Follow-up analysis showed that athletes showing oedema within the cancellous bone and/or stress fracture on MRI had, within 2 years of analysis, subsequently taken time off training and racing due a tibial stress fracture. CONCLUSIONS: The thinner anterior cortex in SF tibiae is associated with a stress reaction in these triathletes.

Chromatinized Protein Kinase C-θ: Can It Escape the Clutches of NF-κB?

We recently provided the first description of a nuclear mechanism used by Protein Kinase C-theta (PKC-θ) to mediate T cell gene expression. In this mode, PKC-θ tethers to chromatin to form an active nuclear complex by interacting with proteins including RNA polymerase II, the histone kinase MSK-1, the demethylase LSD1, and the adaptor molecule 14-3-3ζ at regulatory regions of inducible immune response genes. Moreover, our genome-wide analysis identified many novel PKC-θ target genes and microRNAs implicated in T cell development, differentiation, apoptosis, and proliferation. We have expanded our ChIP-on-chip analysis and have now identified a transcription factor motif containing NF-κB binding sites that may facilitate recruitment of PKC-θ to chromatin at coding genes. Furthermore, NF-κB association with chromatin appears to be a prerequisite for the assembly of the PKC-θ active complex. In contrast, a distinct NF-κB-containing module appears to operate at PKC-θ targeted microRNA genes, and here NF-κB negatively regulates microRNA gene transcription. Our efforts are also focusing on distinguishing between the nuclear and cytoplasmic functions of PKCs to ascertain how these kinases may synergize their roles as both cytoplasmic signaling proteins and their functions on the chromatin template, together enabling rapid induction of eukaryotic genes. We have identified an alternative sequence within PKC-θ that appears to be important for nuclear translocation of this kinase. Understanding the molecular mechanisms used by signal transduction kinases to elicit specific and distinct transcriptional programs in T cells will enable scientists to refine current therapeutic strategies for autoimmune diseases and cancer.

Chromatin-associated protein kinase C-θ regulates an inducible gene expression program and microRNAs in human T lymphocytes.

Studies in yeast demonstrate that signaling kinases have a surprisingly active role in the nucleus, where they tether to chromatin and modulate gene expression programs. Despite these seminal studies, the nuclear mechanism of how signaling kinases control transcription of mammalian genes is in its infancy. Here, we provide evidence for a hitherto unknown function of protein kinase C-theta (PKC-θ), which physically associates with the regulatory regions of inducible immune response genes in human T cells. Chromatin-anchored PKC-θ forms an active nuclear complex by interacting with RNA polymerase II, the histone kinase MSK-1, and the adaptor molecule 14-3-3ζ. ChIP-on-chip reveals that PKC-θ binds to promoters and transcribed regions of genes, as well as to microRNA promoters that are crucial for cytokine regulation. Our results provide a molecular explanation for the role of PKC-θ not only in normal T cell function, but also in circumstances of its ectopic expression in cancer.

The interaction between skill, postures, forces and back pain in wool handling.

Wool handling is an important rural occupation where workers process 200 or more fleeces daily, separating them into various quality components. Loads and postures they experience carry substantial risk of low back pain (LBP). Although a formal skill training structure exists, interaction with loads and LBP is unknown. We examined whether skill and LBP influenced trunk postures and loads of 60 wool handlers representing 3 skill levels. LBP prevalence ranged from 20% for junior (lowest skill) to 45% for open class (highest skill) wool handlers. Open class wool handlers demonstrated increased lateral bend and more axially twisted postures, generating greater medio-lateral shear forces and lateral bend and axial twist moments. LBP was associated with open class wool handlers spending more time in severe axially twisted postures. These findings suggest that skill-based training needs to be reviewed to reduce the quantity of axially twisted posture which may help reduce the prevalence of LBP in this workforce.

Cognitive performance and leukocyte telomere length in two narrow age-range cohorts: a population study.

BACKGROUND: Cognitive function and telomere length both decline with age. A correlation between these two measures would suggest that they may be influenced by the same underlying age-related biological process. Several studies suggest telomere length may be positively correlated with cognitive performance but the evidence is equivocal. In this report, the relationships between telomere length and cognitive performance at Wave 2 and cognitive change from Wave 1 to Wave 2 are assessed in two narrow age-range population cohorts. METHODS: We tested the hypothesis that leukocyte telomere length correlates positively with cognitive performance and cognitive decline in two community cohorts of middle-aged (n = 351, 44-49 years) and older (n = 295, 64-70 years) adults, who participated in two waves of a longitudinal study undertaken in the Canberra-Queanbeyan region of Australia. Telomere length was estimated at Wave 2. Cognitive performance was measured using the Symbol Digit Modalities Test, the immediate recall test of the California Verbal Learning Test, reaction time (simple & choice) and the Trails Test Part B. RESULTS: Cross-sectionally at Wave 2, telomere length correlated with Symbol Digit Modalities Test scores (men) and simple reaction time (women) for the older cohort only, although the latter finding was in the opposite direction to that hypothesised. Telomere length measured at Wave 2 was not associated with cognitive change from Wave 1 to Wave 2 for either cohort, except for two associations of small magnitude (immediate recall in the older cohort, choice reaction time in older women), which were also in the contrary direction to that predicted. CONCLUSIONS: These results do not give strong support to the hypothesis that leukocyte telomere length is associated with either levels of cognitive performance or age-related cognitive change.

No associations between telomere length and age-sensitive indicators of physical function in mid and later life.

Telomere length, which declines with age, has been hypothesized to act as an indicator of biological aging. If it fulfills this purpose, shorter telomere length should correlate with age-related loss of physical function, independent of age. In this cross-sectional Australian population study, the associations between peripheral blood leukocyte telomere length and age-sensitive indicators of physical function (lung function, blood pressure, and grip strength) were examined in two narrow age range cohorts aged 44-49 years (n = 351) and 64-70 years (n = 295). Telomere length was correlated with systolic blood pressure but only for women of the younger cohort and in the opposite direction to that expected (partial r = .181, p = .017). This evidence does not provide support for the hypothesis that telomere length is related to age-associated changes in physical function.

Match and training injuries in rugby league: a review of published studies.

Rugby league is an international collision sport played by junior, amateur, semiprofessional and professional players. The game requires participants to be involved in physically demanding activities such as running, tackling, passing and sprinting, and musculoskeletal injuries are common. A review of injuries in junior and senior rugby league players published in Sports Medicine in 2004 reported that injuries to the head and neck and muscular injuries were common in senior rugby league players, while fractures and injuries to the knee were common in junior players. This current review updates the descriptive data on rugby league epidemiology and adds information for semiprofessional, amateur and junior levels of participation in both match and training environments using studies identified through searches of PubMed, CINHAL, Ovid, MEDLINE, SCOPUS and SportDiscus databases. This review also discusses the issues surrounding the definitions of injury exposure, injury rate, injury severity and classification of injury site and type for rugby league injuries. Studies on the incidence of injuries in rugby league have suffered from inconsistencies in the injury definitions utilized. Some studies on rugby league injuries have utilized a criterion of a missed matchas an injury definition, total injury incidences or a combination of both time-loss and non-time-loss injuries, while other studies have incorporated a medical treatment injury definition. Efforts to establish a standard definition for rugby league injuries have been difficult, especially as some researchers were not in favour of a definition that was all-encompassing and enabled non-time-loss injuries to be recorded. A definition of rugby league injury has been suggested based on agreement by a group of international researchers. The majority of injuries occur in the match environment, with rates typically increasing as the playing level increases. However, professional level injury rates were reportedly less than semiprofessional participation. Only a few studies have reported training injuries in rugby league, where injury rates were reported to be less than match injuries. Approximately 16-30% of all rugby league injuries have been reported as severe, which places demands upon other team members and, if the player returns to playing too early, places them at an increased risk of further injuries. Early research in rugby league identified that ligament and joint injuries were the common injuries, occurring primarily to the knee. More recently, studies have shown a change in anatomical injury sites at all levels of participation. Although the lower limb was the frequent injury region reported previously, the shoulder has now been reported to be the most common injury site. Changes in injury site and type could be used to prompt further research and development of injury reduction programmes to readdress the issue of injuries that occur as a result of participation in rugby league activities. Further research is warranted at all participation levels of rugby league in both the match and training environments to confirm the strongest risk factors for injury.

Dynamic histone variant exchange accompanies gene induction in T cells.

Changes in chromatin composition are often a prerequisite for gene induction. Nonallelic histone variants have recently emerged as key players in transcriptional control and chromatin modulation. While the changes in chromatin accessibility and histone posttranslational modification (PTM) distribution that accompany gene induction are well documented, the dynamics of histone variant exchange that parallel these events are still poorly defined. In this study, we have examined the changes in histone variant distribution that accompany activation of the inducible CD69 and heparanase genes in T cells. We demonstrate that the chromatin accessibility increases that accompany the induction of both of these genes are not associated with nucleosome loss but instead are paralleled by changes in histone variant distribution. Specifically, induction of these genes was paralleled by depletion of the H2A.Z histone variant and concomitant deposition of H3.3. Furthermore, H3.3 deposition was accompanied by changes in PTM patterns consistent with H3.3 enriching or depleting different PTMs upon incorporation into chromatin. Nevertheless, we present evidence that these H3.3-borne PTMs can be negated by recruited enzymatic activities. From these observations, we propose that H3.3 deposition may both facilitate chromatin accessibility increases by destabilizing nucleosomes and compete with recruited histone modifiers to alter PTM patterns upon gene induction.

Antibiotic ointment in the treatment of Grover disease.

Grover disease, or transient acantholytic dermatosis, chiefly affects the upper part of the trunk in men older than 40 years. Lesions may last for weeks, months, or years, and often are accompanied by intense pruritus. Some patients respond to topical steroid treatment but many do not. This article reports major or total resolution of Grover disease in 6 of 9 patients following topical application of a triple antibiotic ointment. It also proposes using a case registry as a way of further investigating the efficacy of this treatment so that dermatologists may participate.

Association of ground hardness with injuries in rugby union.

BACKGROUND: Ground hardness is considered one of the possible risk factors associated with rugby injuries. OBJECTIVES: To examine the contribution of ground hardness, rainfall and evapotranspiration to the incidence of injury, and to investigate seasonal injury bias throughout one full season of rugby union. METHODS: A prospective epidemiological study of rugby injuries was performed on 271 players from rugby union teams involved in the premier grade rugby competition in Dunedin, New Zealand. Ground hardness was measured before each match over 20 rounds with an industrial penetrometer, and local weather information was collected through the National Institute of Weather and Atmospheric Research and the Otago Regional Council. Poisson mixed models were used to describe injury incidence as a function of ground hardness throughout the season. RESULTS: The overall injury incidence during the season was 52 injuries per 1000 match player-hours (95% CI 42 to 65). Although injury incidence decreased gradually by round with a rate ratio of 0.98 (95% CI 0.96 to 0.99) (p = 0.036), and the hardness of match grounds decreased significantly over the season (0.16 MPa/round, 95% CI 0.12 to 0.21, p<0.001), a non-significant association was demonstrated between injury incidence and ground hardness. Injury incidence was not associated with a combination of ground hardness, rainfall and evapotranspiration on the day of the match or cumulative rainfall and evapotranspiration before each match. CONCLUSIONS: Seasonal change in ground hardness and an early-season bias of injuries was demonstrated. Although the contribution of ground hardness to injury incidence was not statistically significant, match round and injury incidence were highly correlated, confirming a seasonal bias, which may confound the relationship of injury to ground condition.