Human Cytomegalovirus Infection Leads to Elevated Levels of Transplant Arteriosclerosis in a Humanized Mouse Aortic Xenograft Model

Recent findings emphasized an important role of human cytomegalovirus (HCMV) infection in the development of transplant arteriosclerosis. Therefore, the aim of this study was to develop a human peripheral blood lymphocyte (hu‐PBL)/Rag‐2^–/–γc^–/– mouse‐xenograft‐model to investigate both immunological as well as viral effector mechanisms in the progression of transplant arteriosclerosis. For this, sidebranches from the internal mammary artery were recovered during coronary artery bypass graft surgery, tissue‐typed and infected with HCMV. Then, size‐matched sidebranches were implanted into the infrarenal aorta of Rag‐2^–/–γc^–/– mice. The animals were reconstituted with human peripheral blood mononuclear cells (PBMCs) 7 days after transplantation. HCMV‐infection was confirmed by Taqman‐PCR and immunofluorescence analyses. Arterial grafts were analyzed by histology on day 40 after transplantation. PBMC‐reconstituted Rag‐2^–/–γc^–/– animals showed splenic chimerism levels ranging from 1–16% human cells. After reconstitution, Rag‐2^–/–γc^–/– mice developed human leukocyte infiltrates in their grafts and vascular lesions that were significantly elevated after infection. Cellular infiltration revealed significantly increased ICAM‐1 and PDGF‐R‐β expression after HCMV‐infection of the graft. Arterial grafts from unreconstituted Rag‐2^–/–γc^–/– recipients showed no vascular lesions. These data demonstrate a causative relationship between HCMV‐infection as an isolated risk factor and the development of transplant‐arteriosclerosis in a humanized mouse arterial‐transplant‐model possibly by elevated ICAM‐1 and PDGF‐R‐β expression.     

Humanized C3 Mouse: A Novel Accelerated Model of C3 Glomerulopathy

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Fc‐Disabled Anti‐Mouse CD40L Antibodies Retain Efficacy in Promoting Transplantation Tolerance

CD40L antibodies have proven to be powerful immunosuppressive agents in nonhuman primates but unfortunately perturb blood coagulation. Neither the therapeutic nor the prothrombotic mechanism of anti‐CD40L is defined sufficiently to determine whether these effects can be uncoupled. Recent evidence suggests that the Fc region of anti‐CD40L antibodies interacting with Fc receptors plays an important role in stabilizing platelet aggregates. An Fc‐disabled, aglycosylated anti‐CD40L heavy chain variant was therefore created to determine whether it might still be useful in promoting transplantation tolerance. In a number of mouse models an engineered aglycosyl anti‐CD40L recapitulated the effects of the intact anti‐CD40L antibody in tolerance protocols involving transplantation of allogeneic bone marrow and skin. In contrast, another anti‐CD40L variant with a conventional rat γ2b heavy chain was less effective in ensuring long‐term skin graft survival, possibly associated with its faster clearance from the circulation. These results show that short pulses of anti‐CD40L antibody therapy may still be useful in tolerance protocols even when the Fc region is disabled.     

Human Skin Xenograft Rejection in CD45 Exon-6 Knockout Mice: The Implication of Involvement of a Direct Pathway

The results of previous studies indicate that only CD4⁺ T cells generated via the indirect pathway play an essential role in causing discordant skin xenograft rejection. The present study was conducted in an attempt to clarify further the roles of effector T cells generated via direct pathways on discordant xenograft rejection using CD45 exon-6 knockout (CD45−/−; C57BL/6 (B6): H-2^b) mice. It has been strongly suggested that CD45 exon-6 knockout mice have profound impairment in T-cell functions via an indirect pathway. When human skin was grafted onto untreated normal C57BL/6 (B6; H-2^b) mice, rejection occurred within 12 days; however, in the CD45 exon-6 knockout mice, the grafts lasted for slightly longer as in fully allogeneic C3H (H-2^k) skin rejection, with a mean survival time ± SD of 19.4 ± 1.5 days and median survival times of 19 days. The difference in survival periods between the human and C3H skin grafts in the CD45 knockout mice was not statistically significant. Both CD4⁺ and CD8⁺ T cells seemed activated in the spleens of these CD45 exon-6 knockout mice 10 days after the human skin grafting. These results suggest that effector T cells generated via a direct pathway can cause discordant skin xenograft rejection, and that CD45 exon-6 knockout mice can generate effector T cells via a direct pathway to reject discordant skin xenografts, similarly to fully allogeneic skin allografts. Received: June 24, 1999 / Accepted: May 30, 2000     

Costimulation blockade in pig artery patch xenotransplantation - a simple model to monitor the adaptive immune response in nonhuman primates

Ezzelarab MB, Ekser B, Echeverri G, Hara H, Ezzelarab C, Long C, Bajona P, Garcia B, Murase N, Ayares D, Cooper DKC. Costimulation blockade in pig artery patch xenotransplantation – a simple model to monitor the adaptive immune response in nonhuman primates. Xenotransplantation 2012; 19: 221–232. © 2012 John Wiley & Sons A/S. Abstract: Background: CD154 blockade‐based immunosuppression successfully prevents both humoral and cellular adaptive immune responses in baboons receiving α1,3‐galactosyltransferase gene‐knockout (GTKO) pig organs. Using a GTKO pig artery transplantation model in baboons, we evaluated the efficacy of CD28/B7 costimulatory pathway blockade in comparison with CD154 blockade. Methods: Baboons received artery patch grafts from GTKO pigs, with no (Group1), anti‐CD154mAb‐based (Group2), or CTLA4‐Ig‐based (Group3) immunosuppressive therapy. Anti‐pig IgM and IgG antibody and cellular responses were monitored. Xenografts were immunohistologically evaluated for antibody and complement deposition, and cellular infiltration. Results: Group1 baboons developed increased IgM and IgG antibody and cellular responses against GTKO antigens. In Group2, anti‐CD154mAb alone prevented the development of both IgM and IgG antibody and cellular responses,but not cellular infiltration of the graft. In the single baboon that received anti‐thymocyte globulin (ATG) + mycophenolate mofetil (MMF) + anti‐CD154mAb, cellular infiltration of the graft was not seen. In Group3, CTLA4‐Ig with ATG + MMF inhibited the cellular proliferative response to pig antigens but did not prevent the IgG response or cellular infiltration. Conclusions: (i) Artery patch transplantation is a simple model to monitor the adaptive immune response to xenografts; (ii) anti‐CD154mAb prevents sensitization but not cellular infiltration (but, without anticoagulation, may result in early thrombosis of a pig xenograft); (iii) although in only one baboon, the addition of ATG and MMF prevents cellular infiltration and (iv) replacement of anti‐CD154mAb by CTLA4‐Ig (at the doses used), even in combination with ATG and MMF, prevents the cellular proliferative response to GTKO pig antigens but is insufficient to prevent the development of anti‐pig antibodies.     

Immunosuppression for delayed or slow graft function in primary cadaveric renal transplantation: use of low dose tacrolimus therapy with post-operative administration of anti-CD25 monoclonal antibody

BACKGROUND: Patients who develop delayed graft function (DGF) following cadaveric renal transplantation have inferior survival to those who do not. Calcineurin inhibitors (CNI) may prolong recovery from DGF. Patients with DGF are therefore routinely treated with either polyclonal antilymphocyte preparations or monoclonal anti-CD3 monoclonal antibodies and delayed introduction of CNI. The purpose of this study was to evaluate the efficacy of the anti-CD25 monoclonal antibody basiliximab (BSLIX) started post-operatively in patients at high risk for DGF combined with low dose tacrolimus (TAC). METHODS: Patients who received a primary cadaveric renal transplant only after August 1998 were included in this retrospective study (n = 143). All patients received TAC and mycophenolate Mofetil (MMF) pre-operatively. At 6 h post-operatively, graft function was assessed clinically by urine output and serum creatinine. Those patients who had a urine output < 300 cc/6 h or a rising serum creatinine were presumed to be at risk for DGF (n = 46). These patients were treated with 20 mg BSLIX and had TAC dose reduced to maintain a trough blood level of < 5 ng/mL. Basiliximab was repeated at day 5. Patients not felt to be at risk for DGF were treated with standard TAC dose with trough level target of 9-12 ng/mL. Patients at risk were classified as DGF if they needed dialysis or as slow graft function (SGF) if they did not. The combined group (SGF/DGF) were analysed together. Patients with SGF/DGF had their TAC dose increased to achieve trough levels of 9-12 ng/mL when renal function improved. Patient groups were compared for demographics, need for dialysis, serum creatinine, glomerular filtration rate (GFR), TAC trough levels, MMF dosage, complications and 1- and 2-yr actuarial graft survival. RESULTS: Patients with SGF/DGF had a longer length of stay (8 vs. 5.7 d), were more likely to be black (41.3 vs. 25.7%), and required more post-operative haemodialysis (HD) (52.2 vs. 4.1%). SGF/DGF and non-SGF/DGF patients had similar rates of rejection (28.2 vs. 19.6%, p = 0.28) and steroid resistant rejection (SRR) (6.5 vs. 2.1%, p = 0.32). There were no differences in the rate of cytomegalovirus (CMV) infection (4.3 vs. 6.1%). Serum creatinine was higher and GFR lower at all time points in the SGF/DGF patients. The 1 and 2 yr actuarial survival in the non-SGF/DGF patients was 97.6 and 97.6% compared with 1 and 2 yrs actuarial survival of 94.1% and 80.0% in the SGF/DGF patients, p = 0.04. There were no differences in patient survival. There were no differences in actuarial survival for the SGF/DGF patients who received dialysis compared with those who did not receive dialysis. Comparison of patients who received HD (n = 28) to those who did not (n = 115), regardless of group demonstrated no difference in 1 and 2 yrs actuarial survival, 100 and 94.1% in HD patients vs. 98.2 and 92.5% in non-HD patients. CONCLUSIONS: The clinical diagnosis of SGF/DGF can be made 6 h post-operatively based on urine output and serum creatinine. Basiliximab can be started post-operatively in these patients and decreased levels of TAC can be used to achieve acceptably low rates of rejection in these patients. However, SGF/DGF patients, regardless of their need for dialysis, have worse function at 1 yr and lower 2-yr actuarial graft survival compared with non-SGF/DGF patients. Most of the poor survival can be attributed to the SGF group. Further strategies to either prevent SGF/DGF or to optimize treatment in these patients are needed.     

The low dose of rituximab in ABO-incompatible kidney transplantation without a splenectomy: a single-center experience

Shirakawa H, Ishida H, Shimizu T, Omoto K, Iida S, Toki D, Tanabe K. The low dose of rituximab in ABO‐incompatible kidney transplantation without a splenectomy: a single‐center experience.
Clin Transplant 2011: 25: 878–884. © 2010 John Wiley & Sons A/S. Abstract: Purpose: A new protocol for ABO‐incompatible (ABO‐i) kidney transplantation including rituximab was introduced in January 2005 in our institute. This study reviewed the results and evaluated the use of low‐dose rituximab in ABO‐i kidney transplantation. Material and methods: Seventy‐four de novo ABO‐i kidney transplantations were performed at Tokyo Women’s Medical University between January 2005 and August 2010. The immunosuppressive protocol was consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone. All the patients received induction therapy with basiliximab. The pre‐conditioning protocol included double‐filtration plasmapheresis and a single dose of rituximab. A dose of 500 mg/body rituximab was initially employed and yielded excellent results (Group I, n = 24). Afterward, the dose of rituximab was reduced to 200 mg/body in January 2007 (Group II, n = 50). Results: Seventy‐four de novo ABO‐i recipients were treated with this protocol, and all patients underwent kidney transplantation successfully. Effective elimination of the peripheral blood CD19 cells was observed in both groups. However, the peripheral blood CD19 levels were still low in both groups at 24 months after treatment. Conclusion: The patients in Group II showed excellent results similar to Group I. These results suggest that the low dose of rituximab (200 mg/body) is the sufficient dose in ABO‐i kidney transplantation.     

Transplant Glomerulopathy: Ultrastructural Abnormalities Occur Early in Longitudinal Analysis of Protocol Biopsies

Transplant glomerulopathy (TXG) presents a distinctive pattern of glomerular abnormalities. The aim of this study was to describe its sequential ultrastructural pathology. A paired cohort study of 228 protocol biopsies, from our longitudinal database (n = 1345), compared TXG (7 patients, 95 biopsies) and controls (8 patients, 133 biopsies). Ultrastructural morphometry and C4d immunoperoxidase were evaluated from implantation to 5 years after transplantation against sequential histology and functional changes. TXG was predated by early glomerular endothelial cell activation; typified by vacuolation, hypertrophy, serration and expansion of lamina rara interna from 39 +/- 23 days after transplantation. Endothelial cells were transformed into an activated phenotype, containing numerous mitochondria, Golgi and ribosomes. Transition from fenestrated to continuous endothelium, mesangial matrix expansion and podocyte fusion occurred late. Endothelial cell activation also occurred in peritubular capillaries (PTC) followed by basement membrane multi-lamination (p < 0.05-0.001). Light microscopy changes of TXG occurred at 2.3 years. PTC C4d deposition was intermittently expressed over time, correlating with endothelial abnormalities, glomerular C4d and donor-specific antibodies (DSA) (p < 0.05-0.001). In summary, endothelial and subendothelial ultrastructural abnormalities in glomerular and peritubular capillaries are sensitive, early markers of TXG, likely due to stimulation of endothelial cells into an activated phenotype by antibody-mediated sub-lytic complement deposition.     

Improved Hepatic Regeneration With Reduced Injury by Redox Factor-1 in a Rat Small-Sized Liver Transplant Model

Redox factor-1 (Ref-1) has been shown to function in a redox-dependent manner in the cell. This study was designed to examine the effects of Ref-1 on liver regeneration as well as protection against postischemic injury in a rat model of 20% partial liver transplantation. Adenovirus carrying the full length of Ref-1 gene was introduced into liver grafts by ex vivo perfusion via the portal vein during preservation. Liver graft weights were assessed, as well as graft histology, serum levels of alanine aminotransferase (ALT)/bilirubin, DNA binding activities of AP-1 and Stat3. Redox factor-1 successfully expressed in the liver graft, improved regeneration by promoting cell proliferation. Overexpression of Ref-1 protein also reduced post-transplant injury and inflammatory reactions in the grafts. The increased serum levels of ALT and bilirubin observed after transplantation were significantly reduced by Ref-1 overexpression. Furthermore, adenovirally overexpressed Ref-1 in mouse liver successfully promoted liver regeneration after simple partial hepatectomy. Interestingly, Ref-1 significantly increased DNA binding of Stat3, but not AP-1. Overexpressed Ref-1 effectively promoted graft regeneration and reduced postischemic injury in a small-sized liver transplantation model. The results of the present study may open a new avenue to clinical transplantation of disproportionately sized grafts in living-related liver transplantation.     

Switch to a Sirolimus‐Based Immunosuppression in Long‐Term Renal Transplant Recipients: Reduced Rate of (Pre‐)Malignancies and Nonmelanoma Skin Cancer in a Prospective,Randomized, Assessor‐Blinded,Controlled Clinical Trial

Renal transplant recipients (RTR) have a 50–200‐fold higher risk for nonmelanoma‐skin cancer (NMSC) causing high rates of morbidity and sometimes mortality. Cohort‐studies gave evidence that a sirolimus‐based immunosuppression may inhibit skin tumor growth. This single‐center, prospective, assessor‐blinded, randomized trial investigated if switching to sirolimus treatment inhibits the progression of premalignancies and moreover how many new NMSC occur compared to continuation of the original immunosuppressive therapy. Forty‐four RTR (mean age 59.9 years, mean duration of immunosuppression 229.5 months) with skin lesions were randomized to sirolimus or continuation of their original immunosuppression. Blinded dermatological assessment at month 6 and 12 by the same dermatologist evaluated the clinical change compared to baseline. Biopsy was performed in suspected malignancy. Already the 6‐month‐assessment showed significant superiority of sirolimus‐therapy: a stop of progression, even regression of preexisting premalignancies (p < 0.0005). This effect was increased at month 12 (p < 0.0001). Nine patients developed histologically confirmed NMSC: one in the sirolimus group, eight in the control group, p = 0.0176. Sirolimus‐based immunosuppression in RTR, even when established many years after transplantation, can delay the development of premalignancies, induce regression of preexisting lesions and decelerate the incidence of new NMSC.     

Impact of Mixed Xenogeneic Porcine Hematopoietic Chimerism on Human NK Cell Recognition in a Humanized Mouse Model

Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse natural killer (NK) cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an in vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells but was associated with an alteration in NK cell subset distribution and interferon gamma (IFN‐γ) production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while ensuring adequate NK cell functions.     

Prospective Analysis of Human Cytomegalovirus DNAemia and Specific CD8+ T Cell Responses in Lung Transplant Recipients

In lung transplant recipients (LuTRs), human cytomegalovirus (HCMV) DNAemia may be associated with HCMV disease and reduced survival of the allograft. Because T cells are essential for controlling HCMV replication, we investigated in this prospective study whether the kinetics of plasma HCMV DNA loads in LuTRs are associated with HCMV‐specific CD8+ T cell responses, which were longitudinally assessed using a standardized assay. Sixty‐seven LuTRs were monitored during the first year posttransplantation, with a mean of 17 HCMV DNA PCR quantifications and 11.5 CD8+ T cell tests performed per patient. HCMV‐specific CD8+ T cell responses displayed variable kinetics in different patients, differed significantly before the onset of HCMV DNAemia in LuTRs who subsequently experienced episodes of DNAemia with high (>1000 copies/mL) and low plasma DNA levels (p = 0.0046, Fisher's exact test), and were absent before HCMV disease. In HCMV‐seropositive LuTRs, high‐level DNAemia requiring preemptive therapy occurred more frequently when HCMV‐specific CD8+ T cell responses fluctuated, were detected only after HCMV DNA detection, or remained undetectable (p = 0.0392, Fisher's exact test). Thus, our data indicate that HCMV‐specific CD8+ T cells influence the magnitude of HCMV DNAemia episodes, and we propose that a standardized measurement of CD8+ T cell immunity might contribute to monitoring the immune status of LuTRs posttransplantation.     

Cluster Analysis of Lesions in Nonselected Kidney Transplant Biopsies: Microcirculation Changes,Tubulointerstitial Inflammation and Scarring

Banff classification empirically established scoring of histologic lesions, but the relationships of lesions to each other and to underlying biologic processes remain unclear. We hypothesized that class discovery tools would reveal new relationships between individual lesions, and relate lesions to C4d staining, anti‐HLA donor‐specific antibody (DSA) and time posttransplant. We studied 234 nonselected renal allograft biopsies for clinical indications from 173 patients. Silhouette plotting and principal component analysis revealed three groups of lesions: microcirculation changes, including inflammation (glomerulitis, capillaritis) and deterioration (double contours, mesangial expansion); scarring/hyalinosis; and tubulointerstitial inflammation. DSA and C4d grouped with microcirculation inflammation, whereas time posttransplant grouped with scarring/hyalinosis lesions. Intimal arteritis clustered with DSA, C4d and microcirculation inflammation, but also showed correlations with tubulitis. Fibrous intimal thickening in arteries clustered with scarring/hyalinosis. Capillary basement membrane multilayering showed intermediary relationships between microcirculation deterioration and time‐dependent scarring. Correlation analysis and hierarchical clustering confirmed the lesion relationships. Thus, we propose that the pathologic lesions in biopsies are not independent but are members of groups that represent distinct pathogenic forces: microcirculation changes, reflecting the stress of DSA; scarring, hyalinosis and arterial fibrosis, reflecting the cumulative burden of injury over time; and tubulointerstitial inflammation. Interpretation of lesions should reflect these associations.     

Infection with the Intracellular Bacterium,Listeria monocytogenes,Overrides Established Tolerance in a Mouse Cardiac Allograft Model

Infections and TLR signals at the time of transplantation have been shown to prevent the induction of tolerance, but their effect on allografts after tolerance has been established is unclear. We here report that infection with Listeria monocytogenes precipitated the loss of tolerance and the MyD88‐ and T cell‐dependent rejection of accepted cardiac allografts in mice. This loss of tolerance was associated with increases in the numbers of graft‐infiltrating macrophages and dendritic cells, as well as CD4⁺FoxP3^? and CD8⁺ T cells. Rejection was also associated with increased numbers of graft‐infiltrating alloreactive as well as Listeria‐reactive IFNγ‐producing T cells. Rejection of the established grafts required both IL‐6 and IFNß, cytokines produced during acute Listeria infection. However, IL‐6 and IFNß alone, even when present at higher concentrations than during Listeria infection, were insufficient to break tolerance, while the combination of IL‐6 and IFNß was sufficient to break tolerance. These and in vitro observations that IL‐6 but not IFNß enhanced T cell proliferation while IFNß but not IL‐6 enhanced IFNγ production support a hypothesis that these cytokines play nonredundant roles. In conclusion, these studies demonstrate that the proinflammatory effects of infections can induce the loss of tolerance and acute rejection of accepted allografts.     

Critical role of proinflammatory cytokine IL-6 in allograft rejection and tolerance

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.     

Selective CD28 Blockade Attenuates Acute and Chronic Rejection of Murine Cardiac Allografts in a CTLA‐4‐Dependent Manner

Selective blockade of CD28 is a promising therapy to inhibit pathogenic alloimmunity. However, evaluation of this approach in transplantation has been very limited. Using a novel nonactivating single‐chain Fv‐based reagent (α28scFv), we have investigated the role of CD28 and cytotoxic T lymphocyte antigen 4 (CTLA‐4) in a murine cardiac transplant model. Blockade of CD28 for 2 weeks after engraftment promoted allograft survival, and significantly attenuated chronic rejection when combined with transient CD154‐blockade or calcineurin inhibition. Graft acceptance was associated with decreased alloantibody production, increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Blockade of CTLA‐4 during α28scFv‐based treatments led to prompt rejection in all animals and inhibited expression of forkhead box P3 (Foxp3), programmed death (PD)‐1 and 2,3‐indoleamine dioxygenase (IDO) in the graft. These results show that CD28 signaling during the first weeks after transplant is a pivotal mediator of pathogenic alloimmunity, and that selective CD28 blockade prolongs graft acceptance by at least two immunomodulatory mechanisms. Selective CD28 inhibition while sparing CTLA‐4 is thus a promising approach to inhibit pathogenic alloimmunity.     

Bone Fracture Toughness and Strength Correlate With Collagen Cross‐Link Maturity in a Dose‐Controlled Lathyrism Mouse Model

Collagen cross‐linking is altered in many diseases of bone, and enzymatic collagen cross‐links are important to bone quality, as evidenced by losses of strength after lysyl oxidase inhibition (lathyrism). We hypothesized that cross‐links also contribute directly to bone fracture toughness. A mouse model of lathyrism using subcutaneous injection of up to 500 mg/kg β‐aminopropionitrile (BAPN) was developed and characterized (60 animals across 4 dosage groups). Three weeks of 150 or 350 mg/kg BAPN treatment in young, growing mice significantly reduced cortical bone fracture toughness, strength, and pyridinoline cross‐link content. Ratios reflecting relative cross‐link maturity were positive regressors of fracture toughness (HP/[DHLNL + HLNL] r² = 0.208, p < 0.05; [HP + LP]/[DHNL + HLNL] r² = 0.196, p < 0.1), whereas quantities of mature pyridinoline cross‐links were significant positive regressors of tissue strength (lysyl pyridinoline r² = 0.159, p = 0.014; hydroxylysyl pyridinoline r² = 0.112, p < 0.05). Immature and pyrrole cross‐links, which were not significantly reduced by BAPN, did not correlate with mechanical properties. The effect of BAPN treatment on mechanical properties was dose specific, with the greatest impact found at the intermediate (350 mg/kg) dose. Calcein labeling was used to define locations of new bone formation, allowing for the identification of regions of normally cross‐linked (preexisting) and BAPN‐treated (newly formed, cross‐link‐deficient) bone. Raman spectroscopy revealed spatial differences attributable to relative tissue age and effects of cross‐link inhibition. Newly deposited tissues had lower mineral/matrix, carbonate/phosphate, and Amide I cross‐link (matrix maturity) ratios compared with preexisting tissues. BAPN treatment did not affect mineral measures but significantly increased the cross‐link (matrix maturity) ratio compared with newly formed control tissue. Our study reveals that spatially localized effects of short‐term BAPN cross‐link inhibition can alter the whole‐bone collagen cross‐link profile to a measureable degree, and this cross‐link profile correlates with bone fracture toughness and strength. Thus, cross‐link profile perturbations associated with bone disease may provide insight into bone mechanical quality and fracture risk. © 2014 American Society for Bone and Mineral Research.