Andrés Jaramillo | Mayo Clinic (original) (raw)

Papers by Andrés Jaramillo

Clinical Transplantation, 2024

This single-center retrospective study investigated subclinical rejection prevalence and signific... more This single-center retrospective study investigated subclinical rejection prevalence and significance in simultaneous pancreas and kidney transplant (SPKT) recipients. We analyzed 352 SPKT recipients from July 2003 to April 2022. Our protocol included pancreas allograft surveillance biopsies at 1, 4, and 12months post-transplant. After excluding 153 patients unable to undergo pancreas biopsy, our study cohort comprised 199 recipients. Among the 199 patients with protocol pancreas biopsies, 107 had multiple protocol pancreas biopsies in the first year, totaling 323. Subclinical rejection was identified in 132 episodes (41%). Of these, 72% were Grade 1, 20% were indeterminate, and 8% were Banff Grade 2 or higher. All episodes of subclinical rejection were treated. Rates of pancreas graft loss (10% vs. 7%) and clinical rejection (21% vs. 20%) at 3 years were similar between those with and without subclinical rejection. Subclinical rejection Banff Grade 2 or more was associated with poor pancreas graft survival HR of 5.5 (95% CI: 1.24-24.37, p = 0.025). Of 236 simultaneous protocol kidney and pancreas biopsies, 102 (43%) showed pancreas subclinical rejection, while only 17% had concurrent kidney subclinical rejection. Our findings suggest limited predictive value of pancreatic enzymes and euglycemia in detecting pancreas rejection. Furthermore, poor concordance existed between pancreas and kidney subclinical rejection.

Kidney360, 2024

The Hispanic population of the United States is the second largest racial or ethnic group, compri... more The Hispanic population of the United States is the second largest racial or ethnic group, comprising 18.7% of the population. However, this population is incredibly heterogeneous differing in genetic traits, cultural upbringing, educational backgrounds, and financial status. The impact of this heterogeneity on the prevalence and outcomes of renal disease and kidney transplantation is understudied compared with non-Hispanic White and Black populations. What is known appears to be underrecognized. This review aims to critically assess current medical literature on Hispanic individuals, focusing on etiological factors, disease progression, and outcomes related to CKD and kidney transplantation. By doing so, we aim to underscore key areas for further in-depth investigation.

The Journal of Heart and Lung Transplantation, 2024

Background: Current monitoring after heart transplantation (HT) employs repeated invasive endomyo... more Background: Current monitoring after heart transplantation (HT) employs repeated invasive endomyocardial biopsies (EMB). Although positive EMB confirms rejection, EMB fails to predict impending, sub-clinical, or EMB-negative rejection events. While non-HLA antibodies have emerged as important risk factors for antibody-mediated rejection (AMR) after HT, their use in clinical risk stratification has been limited. A systematic review of the role of non-HLA antibodies in rejection pathologies has potential to guide efforts to overcome deficiencies of EMB in rejection monitoring. Methods: Databases were searched to include studies on non-HLA antibodies in HT recipients. Data collected included: number of patients, type of rejection, non-HLA antigen studied, association of non-HLA antibodies with rejection, and evidence for synergistic interaction between non-HLA antibodies and HLA-DSA responses. Results: A total of 56 studies met the inclusion criteria. Strength of evidence for each non-HLA antibody was evaluated based on the number of articles and patients in support vs. against their role in mediating rejection. Importantly, despite previous intense focus on the role of anti-MHC class I chain-related gene A (MICA) and anti-angiotensin II type I receptor (AT1R) antibodies in HT rejection, evidence for their involvement was equivocal. Conversely, strength of evidence for other non-HLA antibodies supports that differing rejection pathologies are driven by differing non-HLA antibodies. Conclusion: This systematic review underscores the importance of identifying peri-HT non-HLA antibodies. Current evidence supports the role of non-HLA antibodies in all forms of HT rejection. Further investigations are required to define the mechanisms of action of non-HLA antibodies in HT rejection.

Human Immunology, 2023

Background: Immune response to several kidney self-antigens (KSAg) such as Collagen IV (Col-IV), ... more Background: Immune response to several kidney self-antigens (KSAg) such as Collagen IV (Col-IV), Perlecan (PL), and Fibronectin (FN) have been associated with antibody-mediated damage and poor allograft survival. Thus, the aim of this study was to determine if humoral immune responses to KSAg correlates with progression of chronic immune injury (CII) changes at 1 year or 2 years. Methods: Kidney transplant recipients who underwent 1-or 2-year biopsies, with chronic interstitial inflammation (ci > 1) and/or glomerular membrane double contouring (cg > 0) were analyzed with matched controls. Sera were analyzed retrospectively for antibodies against KSAg using ELISA. The presence of antibodies to KSAg were compared at 0, 4, 12, and 24 months using logistic regression. Results: We identified a cohort of 214 kidney transplant recipients. Of these, we identified 33 cases and matched 66 controls. Logistical regression showed an odds ratio of 1 with the confidence interval crossing 1 for the presence of response to KSAg at all the time points. Conclusions: Humoral immune responses to either KSAg alone or in combination with donor-specific anti-HLA antibodies are not associated with progression to CII at 1 and 2 years after kidney transplantation.

Medicine, 2023

The value of the crossmatch test in assessing pretransplant immunological risk is vital for clini... more The value of the crossmatch test in assessing pretransplant immunological risk is vital for clinical decisions, ranging from the indication of the transplant to the guidance of induction protocols and treatment with immunosuppressants. The crossmatch tests in transplantation can be physical or virtual, each with its advantages and limitations. Currently, the virtual crossmatch stands out for its sensitivity and specificity compared to the physical tests. Additionally, the virtual crossmatch can be performed in less time, allowing for a reduction in cold ischemia time. It shows a good correlation with the results of physical tests and does not negatively impact graft survival.
Proper communication between clinicians and the transplant immunology laboratory will lead to a deeper understanding of each patient's immunological profile, better donor-recipient selection, and improved graft survival.

Biomédica, 2022

La presencia de anticuerpos dirigidos contra los antígenos leucocitarios humanos (Human Leukocyte... more La presencia de anticuerpos dirigidos contra los antígenos leucocitarios humanos (Human Leukocyte Antigens, HLA) que se expresan en las células del donante, es uno de los factores de riesgo más importantes asociados con las complicaciones clínicas después del trasplante. La prueba cruzada es una de las pruebas de histocompatibilidad más eficaces para la detección de anticuerpos específicos contra el donante en los receptores de injertos. En los primeros métodos de la prueba cruzada, se utilizaba la citotoxicidad dependiente del complemento, que es útil para detectar dichos anticuerpos responsables del rechazo hiperagudo del injerto, pero carece de la sensibilidad adecuada. Por ello, se desarrollaron métodos de pruebas cruzadas más sensibles, entre ellas, la prueba cruzada por citometría de flujo que hoy se considera el método preferido.
En este artículo se revisa la evolución de la prueba cruzada y los factores más importantes que deben tenerse en cuenta al realizarla y al interpretar los resultados de esta prueba fundamental para la supervivencia a largo plazo del injerto.

Transplantation Proceedings, 2022

There is a lower incidence of antibody-mediated rejection (AMR) after simultaneous liver-kidney t... more There is a lower incidence of antibody-mediated rejection (AMR) after simultaneous liver-kidney transplantation (SLKT) than after kidney-only transplantation. It has been suggested that soluble human leukocyte antigen (sHLA) produced by the liver protects the kidney from AMR. However, this hypothesis has not been tested after SLKT. We present a case of SLKT with 2 donorspecific antibodies (DSAs) (DR53, 12,364 mean fluorescence intensity [MFI]; DQ7, 1253 MFI) that displayed a decrease by day 7 (DR53, 2747 MFI; DQ7, 107 MFI). On day 351, the patient was diagnosed with kidney AMR associated with high levels of DSA (DR53, 18,542 MFI; DQ7, 22,007 MFI) that persisted until day 531. High levels of sHLA-DR/DQ and HLA-DR/DQ-containing exosomes were also detected on day 398. Consequently, the patient underwent treatment with plasmapheresis, intravenous immunoglobulin, prednisone, and rituximab. On day 752, biopsy results were negative for AMR. Moderate levels of DSA (DR53, 9798 MFI; DQ7, 1271 MFI), and baseline levels of sHLA-DR/DQ and HLA-DR/DQ-containing exosomes were observed. Increases in CD4 + CD25 + FOXP3 + regulatory T cell marker−containing exosomes (CD73, programmed death-ligand 1) were observed on day 752 compared to day 398. These data show a direct correlation between sHLA and HLA-containing exosomes and an inverse correlation between tolerance marker−containing exosomes and kidney AMR after SLKT.

Transplantation Direct, 2022

Background. The Banff classification scheme provides a framework for interpreting transplant kidn... more Background. The Banff classification scheme provides a framework for interpreting transplant kidney biopsies and has undergone various updates in the past 2 decades especially related to antibody-mediated rejection. The clinical significance of early glomerulitis seen within 4 mo on protocol biopsies has received limited attention. We hypothesized that early glomerulitis seen on protocol biopsies will lead to significant adverse outcomes as assessed by histopathology and allograft outcome. Methods. A single-center retrospective study of a cohort of patients who underwent protocol biopsies within 4 mo after transplantation with timely follow-up protocol biopsies were assessed. Patients with recurrent glomerulonephritis were excluded. Results. We calculated glomerulitis (g) scores for 2212 biopsy specimens and identified 186 patients with glomerulitis (g > 0) and 2026 patients without glomerulitis (g = 0). The progression to chronic transplant glomerulopathy at 1 and 2 y was higher in patients with g > 0 as compared with g = 0 (year 1, 10.7% versus 2.3% [P < 0.001]‚ respectively; year 2, 17.2% versus 4.3% [P < 0.001], respectively) with no difference in other chronic lesions. The death-censored graft failure rate was higher in patients with g > 0 as compared with g = 0 (hazard ratio, 1.68 [95% CI, 1.07-2.65]; P = 0.02). We did not find any difference in outcomes in glomerulitis group based on donor-specific antibody. Conclusion. Our findings suggest that early glomerulitis (seen within 4 mo after transplantation) may lead to clinically significant long-term changes and thus could be a target for early intervention therapies.

American Journal of Transplantation, 2022

Simultaneous liver-kidney transplant (SLKT) in the presence of anti-human leukocyte antigen (HLA)... more Simultaneous liver-kidney transplant (SLKT) in the presence of anti-human leukocyte antigen (HLA) donor-specific antibodies (DSA) is a well-accepted practice. Herein, we describe the evolution of alloantibodies in a patient who received an SLKT. The pre-SLKT serum sample showed multiple strong DSA. As expected, all DSA cleared in a sample collected 4 days after the SLKT. Because of primary nonfunction of the liver in the SLKT, the patient had a second liver transplant 4 days later. An abrupt increase in DSA levels against the kidney was detected 10 days after the second liver transplant. These DSA were refractory to treatment, and the transplanted kidney was lost due to antibody-mediated rejection (AMR). A detailed study of the HLA epitopes recognized by DSA and, after normalization with 3rd party alloantibodies to address the effect of multiple transfusions and liver allograft neutralization, showed that the elimination of these antibodies depended on the HLA antigens expressed by the transplanted liver cells. The return of DSA after removal of the first transplanted liver was associated with AMR in the transplanted kidney.

American Journal of Transplantation, 2021

A novel coronavirus has had global impact on individual health and health care delivery. In this ... more A novel coronavirus has had global impact on individual health and health care delivery. In this C4 article, contributors discuss various aspects of transplantation including donor and recipient screening, management of infected patients, and prevention of coronavirus disease (COVID). Donor screening with SARS‐CoV‐2 nucleic acid testing (NAT) close to the time of procurement is recommended. Many programs are also screening all potential recipients at the time of admission. The management of COVID has evolved with remdesivir emerging as a new potential option for transplant recipients. Dexamethasone has also shown promise and convalescent plasma is under study. Prevention strategies for transplant candidates and recipients are paramount. Pediatric‐specific issues are also discussed. Strategies for the psychological well‐being of patients and providers are also imperative, in addition to future research priorities for transplantation.

HLA, 2019

Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping... more Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the “gold standard.” However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation.

HLA, 2019

The lymphocyte crossmatch is currently the only cell-based compatibility assay performed by histo... more The lymphocyte crossmatch is currently the only cell-based compatibility assay performed by histocompatibility laboratories for transplant purposes. While in many transplant programs the complement-dependent cytotoxicity crossmatch (CDCXM) remains in use, when available, the flow cytometry crossmatch (FCXM) is the method of choice because of its superior sensitivity and specificity. Unfortunately, the maintenance and cost of a flow cytometer is a considerable limitation for small histocompatibility laboratories. Therefore, in this study, we evaluated the use of the Cellometer Vision CBA image cytometer (Nexcelom Bioscience LLC, Lawrence, Massachusetts) as an alternative instrument to perform the crossmatch assay. The 3-color FCXM protocol was modified into two separate 2-color panel image cytometry crossmatches (IXMs), one for T cells and one for B cells. After initial serum and cell incubation, a cocktail consisting of PE/Cy5-conjugated anti-human CD3 or CD19 and PE-conjugated anti-human IgG F(ab')2 was added to the T cell and B cell panels, respectively. The final cell preparation was added to a separate counting chamber. Images were captured using the Cellometer Vision CBA, an image cytometer designed for cell counting, size analysis and fluorescence intensity measurement. Thirty-nine IXMs were performed and compared with the FCXM. We obtained a concordance sensitivity of 94.1% and 100% and specificity of 100% and 88.9% for T cells and B cells, respectively. The linearity of the system was verified using dilutions of a sample containing known donor-specific anti-HLA antibodies (DSA) against the target cells. This feasibility study demonstrates that the FCXM test could be easily adapted to the Cellometer Vision CBA image cytometer without compromising specificity and sensitivity. The low instrumentation cost, minimal maintenance, and simple operation allow for efficient implementation or transition from the FCXM to the IXM method.

Clinics in Laboratory Medicine, 2018

The presence of antibodies directed against HLA molecules expressed on the donor&amp;#39;... more The presence of antibodies directed against HLA molecules expressed on the donor&amp;#39;s cells is one the most important risk factor for serious clinical complications after transplantation. The lymphocyte crossmatch is one of the most important tests available to the laboratory as this assay detects the presence of donor-specific anti-HLA antibodies in potential allograft recipients. Early crossmatch methods used a complement-dependent cytotoxicity test, which was useful for detecting anti-HLA antibodies responsible for hyperacute graft rejection but lacked adequate sensitivity and specificity. Consequently, more sensitive and specific crossmatch methods were developed ultimately leading to the flow cytometry crossmatch as the preferred methodology.

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, Jan 27, 2018

Should we continue to let complement-dependent cytotoxicity (CDC) crossmatches deny organs for se... more Should we continue to let complement-dependent cytotoxicity (CDC) crossmatches deny organs for sensitized patients? Or can we safely manage or prevent the potential complications associated with transplanting across a positive crossmatch? In the adult population, there are numerous studies showing poor clinical outcomes so that doing so, for the moment, is not standard of care. This article is protected by copyright. All rights reserved.

Transplant infectious disease : an official journal of the Transplantation Society, 2018

Before the 2014 policy change pertaining to infectious disease screening, many organ procurement ... more Before the 2014 policy change pertaining to infectious disease screening, many organ procurement organizations (OPOs) were supplementing serologic screening of deceased organ donors with nucleic acid testing (NAT) for human immunodeficiency virus (HIV-1), hepatitis B virus (HBV), and hepatitis C virus (HCV). The number of seronegative, NAT-positive donors has not been directly measured. HIV, HBV, and HCV screening results of 11 229 donor referrals evaluated from 2010 to 2013 were obtained from 3 OPO-affiliated laboratories, capturing 35% of all donors in the United States. Laboratories used either polymerase chain reaction assay or transcription-mediated amplification assay to test 9643 deceased donors by NAT. The NAT results were positive in 21 (0.2%), 1 (0.02%), and 11 (0.1%) donors who were seronegative for HIV, HBV, and HCV, respectively. All discordant HIV-1 results were from one laboratory using a polymrease chain reaction assay. Thirteen of the reactive HIV NAT results in ser...

The Journal of sports medicine and physical fitness, Jan 29, 2017

Exercise-induced stress induces considerable changes in the immune system. To better understand t... more Exercise-induced stress induces considerable changes in the immune system. To better understand the mechanisms related to these immune changes during acute and chronic physical stress, we studied the effects of aerobic physical training (APT) on several parameters of the immune system. Previously untrained males (18-25 years of age) were divided into a group that was subjected to 6 months of APT (n=10) and a sedentary control group (n=7). The subjects performed a cardiopulmonary exercise test (CET) at 0, 3, and 6 months of the APT program. B cell (CD19+), T cell (CD4+ and CD8+), and natural killer cell (CD56+) levels, and mitogen-induced T cell proliferation and cytokine production (interleukin-1, interleukin-4, interleukin-12, and interferon-) were evaluated before and at 30 seconds and 24 hours after the CET. There was a significant increase in CD4+ T cells and natural killer cells and a significant reduction in T cell proliferation in both groups 30 seconds after the CET at 3 and...

American Journal of Transplantation

The new national Kidney Allocation System of the Organ Procurement and Transplantation Network (O... more The new national Kidney Allocation System of the Organ Procurement and Transplantation Network (OPTN), effective as of December 4, 2014, was designed to improve the chances of transplanting the most highly sensitized patients on the waitlist, those with calculated panel reactive antibody values of 98%, 99% and 100%. Recently, it was suggested that these highly sensitized patients will experience inequitable access, given the reported high prevalence of antibodies to HLA‐DP, and the fact that only about 1/3 of deceased donors are typed for HLA‐DP antigens. Here we report that 320/2948 flow cytometric crossmatches performed for the Northwestern transplant program over the past 28 months were positive solely due to HLA‐DP donor‐specific antibodies (11%; 16.5% of patients with HLA antibodies—sensitized patients). We further show that 58/207 (12%) HLA‐DR serologically matched donor‐recipient pairs had a positive B cell flow crossmatch due to donor‐specific HLA class II antibodies, and 2/34 (6%) serologic zero‐HLA‐A‐B‐DR mismatch had a positive flow crossmatch due to HLA‐DSA. We therefore provide information regarding the necessity and importance of complete donor HLA typing including both chains of the HLA‐DP antigen (encoded by HLA‐DPA1 and HLA‐DPB1) at the time of organ offer.

Human immunology, Jan 16, 2016

Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody... more Pronase treatment is used in the flow cytometry crossmatch (FCXM) to prevent nonspecific antibody binding on B cells. However, we have observed unexpected positive results with pronase-treated T cells in human immunodeficiency virus (HIV)-infected patients. In this study, 25 HIV-infected patients without HLA antibodies were tested with pronase-treated and nontreated cells. HIV-positive sera were pretreated with reducing agents and preabsorbed with pronase-treated and nontreated T or B cells before crossmatching. All patients displayed FCXM reactivity with pronase-treated T cells but not with nontreated T cells. None of the patients exhibited FCXM reactivity with pronase-treated and nontreated B cells. These patients displayed FCXM reactivity with pronase-treated CD4+ and CD8+ T cells but not with their nontreated counterparts. Preabsorption with pronase-treated T cells reduced the T cell FCXM reactivity. Preabsorption with pronase-treated B cells or nontreated T and B cells did not ...

Clinical Transplantation, 2024

Kidney360, 2024

The Journal of Heart and Lung Transplantation, 2024

Human Immunology, 2023

Medicine, 2023

Biomédica, 2022

Transplantation Proceedings, 2022

Transplantation Direct, 2022

American Journal of Transplantation, 2022

American Journal of Transplantation, 2021

HLA, 2019

Clinics in Laboratory Medicine, 2018

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, Jan 27, 2018

Transplant infectious disease : an official journal of the Transplantation Society, 2018

The Journal of sports medicine and physical fitness, Jan 29, 2017

American Journal of Transplantation

Human immunology, Jan 16, 2016

Human Leukocyte Antigens - Updates and Advances, 2023

The major histocompatibility complex in humans, known as the human leukocyte antigen (HLA) and lo... more The major histocompatibility complex in humans, known as the human leukocyte antigen (HLA) and located on chromosome 6, is the most polymorphic genetic system in humans. The biological role of the HLA class I and class II molecules is to present processed peptides to CD8+ and CD4+ T lymphocytes, respectively. These cells can also respond to foreign (allogeneic) HLA molecules (direct allorecognition) or to foreign HLA-derived peptides (indirect allo-recognition), respectively. Thus, the HLA system controls the acceptance or rejection of transplanted foreign tissues and organs (allografts). High-resolution HLA typing is routinely performed to provide HLA matching in hematopoietic stem cell transplantation to prevent allograft rejection and graft-versus-host disease. In contrast, low-resolution HLA typing is routinely performed in solid organ transplantation to provide HLA matching but, most importantly, to allow for the detection of donor-specific antibodies to prevent antibody-mediated allograft rejection. The capability to amplify DNA by polymerase chain reaction has facilitated the clinical application of molecular techniques and, currently, several molecular HLA typing methods are now available in the histocompatibility laboratory. Herein, we describe the different molecular HLA typing techniques and the different levels of HLA typing resolution used for clinical purposes.

Inmunología: Una Ciencia Activa, 2009

Samter’s Immunologic Diseases, 2001

Human organ transplantation, a combined achievement of medicine, pharmacology, and immunology, pr... more Human organ transplantation, a combined achievement of medicine, pharmacology, and immunology, provides life-saving organs to patients with end-stage organ failure who have exhausted all other therapeutic options. The complexity of the transplant process arises from the immune response to the transplanted organ (rejection), when this organ is derived from another genetically nonidentical individual of the same species (allograft). Allograft rejection is a unique immunologic disease because the onset (transplant procedure) and many of the target antigens (donor antigens) can be easily identified. Despite these opportunities, the most basic aspects of the disease still are not well understood: the nature of the immunogenic peptides (epitopes), the identity of the effector immune mechanisms, and the factors responsible for recipient differences in the immune response to allografts. Therefore, although important advances have been made in the prevention, diagnosis, and treatment protocols, allograft rejection still occurs frequently, is incompletely controlled by current treatment protocols, and represents a major treatment cost.

Immunobiology of Organ Transplantation, 2004

Handbook of Human Immunology, 2008

Introduction to Flow Cytometry, 2019

The presence of antibodies directed against human leucocyte antigen (HLA) molecules expressed on ... more The presence of antibodies directed against human leucocyte antigen (HLA) molecules expressed on the donor’s cells is one the most important risk factor for serious clinical complications after transplantation. Therefore, the crossmatch is one of the most important tests available to the laboratory today as this assay detects the presence of donor-specific anti-HLA antibodies in potential allograft recipients. Early crossmatch methods utilized a complement-dependent cytotoxicity test which was useful for detecting anti-HLA antibodies responsible for hyper-acute graft rejection but lacked adequate sensitivity and specificity. Consequently, more sensitive and specific crossmatch methods were developed ultimately leading to the flow cytometry crossmatch as the current preferred methodology. We review herein the evolution of the crossmatch assay and the important factors to take into consideration while performing, and interpreting results of this fundamental assay for the fate of the transplanted organ.