Introduction Approximately 30% of patients with primary membranous nephropathy (MN) and persistent nephrotic syndrome (NS) fail rituximab therapy through mechanisms that could be overcome by obinutuzumab. Methods In this prospective, single-arm, single-center, open-label trial, twenty consenting adults with MN and rituximab-resistant NS received three 1000 mg obinutuzumab infusions at the Bergamo Nephrology Unit (Italy) between March 2022 and February 2024 and were monitored for at least 12 months. The primary outcome was a composite endpoint of normo-albuminemia and complete (proteinuria <0.3 g/day) or partial (proteinuria <3.5 g/day with ≥50% reduction from baseline) NS remission at 12-month follow-up. Twenty-four-hour proteinuria and GFR were evaluated at baseline and at 3, 6, 9, 12, 18, and 24 months post-treatment. Results At 12 months, 16 patients met the combined endpoint, 4 with complete remission. No patient relapsed after remission. Median [IQR] 24-hour proteinuria decreased from 5.7 [4.7-8.1] to 1.3 [0.5-2.7] g/24-hour. Albumin and IgG fractional clearances also decreased, whereas serum albumin increased from 2.9±0.6 g/dl to 3.9±0.3 g/dl (p<0.0001 for all changes). Dyslipidemia, hypocalcemia and hypo-gammaglobulinemia improved significantly and GFR stabilized. At 12 months, total B-cells and circulating anti-PLA2R antibodies were depleted. Similar findings were observed in the cohort of ten patients who completed 24 months of follow-up. However, total B-cells re-emerged in the circulation without an increase in anti-PLA2R antibodies and proteinuria, or relapses. Treatment was safe and well tolerated. Conclusions Obinutuzumab treatment is extremely effective and safe in patients with MN and rituximab-resistant NS and can achieve persistent remission in this population.

Introduction:Approximately 30% of patients with primary membranous nephropathy (MN) and persistent nephrotic syndrome (NS) fail rituximab therapy through mechanisms that could be overcome by obinutuzumab. Methods:In this prospective, single-arm, single-center, open-label trial, 20 consenting adults with MN and rituximab-resistant NS received three 1000 mg obinutuzumab infusions at the Bergamo Nephrology Unit (Italy) between March 2022 and February 2024 and were monitored for ≤ 12 months. The primary outcome was a composite end point of normo-albuminemia and complete (proteinuria < 0.3 g/d) or partial (proteinuria < 3.5 g/d with ≥ 50% reduction from baseline) NS remission at 12-month follow-up. Twenty-four-hour proteinuria and glomerular filtration rate (GFR) were evaluated at baseline and at 3, 6, 9, 12, 18, and 24 months posttreatment. Results:At 12 months, 16 patients met the combined end point, 4 with complete remission. No patient relapsed after remission. Median (interquartile range) 24-h proteinuria decreased from 5.7 (4.7-8.1) to 1.3 (0.5-2.7) g/24 h. Albumin and IgG fractional clearances also decreased, whereas serum albumin increased from 2.9 ± 0.6 g/dl to 3.9 ± 0.3 g/dl (P < 0.0001 for all changes). Dyslipidemia, hypocalcemia, and hypo-gammaglobulinemia improved significantly and GFR stabilized. At 12 months, total B cells and circulating anti-phospholipase A2 receptor (PLA2R) antibodies were depleted. Similar findings were observed in the cohort of 10 patients who completed 24 months of follow-up. However, total B cells reemerged in the circulation without an increase in anti-PLA2R antibodies and proteinuria, or relapses. Treatment was safe and well-tolerated. Conclusion:Obinutuzumab treatment is extremely effective and safe in patients with MN and rituximab-resistant NS and can achieve persistent remission in this population.

Implementing shortened one-compartment iohexol plasma clearance models for GFR measurement is crucial since the gold standard inulin renal clearance technique and the reference two-compartment, 10-hour, 16-samplings iohexol plasma clearance method are clinically unfeasible. Inulin may precipitate anaphylactic shock. Four-hour and 8-hour one-compartment iohexol plasma clearance models with Bröchner-Mortensen correction provide accurate GFR measurements in patients with estimated GFR (eGFR) > or ≤40 mL/min/1.73m2, respectively. We compared the performance of the simplified 5-hour, 4-samplings, two-compartment population pharmacokinetic model (popPK) with the performance of the reference two-compartment 10-hour iohexol method in 16 patients with GFR 15.2 to 56.5 mL/min/1.73 m2. We also compared the performance of shortened (5, 6 and 7-hour) one-compartment models with the performance of the standard 8-hour one-compartment model in 101 patients with eGFR ≤40 mL/min/1.73 m2. The performance of popPK and shortened methods versus reference methods was evaluated by total deviation index (TDI), concordance correlation coefficient (CCC) and coverage probability (CP). TDI <10%, CCC ≥0.9 and CP >90% indicated adequate performance. TDI, CCC and CP of popPK were 11.11%, 0.809 and 54.10%, respectively. All shortened, one-compartment models overestimated the GFR (p <0.0001 for all) as compared to the 8-hour model. TDI, CCC and CP were 7.02%, 0.815, and 75.80% for the 7-hour model, 7.26%, 0.803, and 74.20% for the 6-hour model, and 8.85%, 0.729 and 64.70% for the 5-hour model. The agreement of popPK model was comparable to that obtained with the Chronic-Kidney-Disease-Collaboration-Epidemiology (CKD-Epi) and the Modification-of-Diet-in-Renal-Disease (MDRD) serum-creatinine based equations for GFR estimation. PopPK model is remarkably unreliable for GFR measurement in stage III-IV CKD patients. In patients with eGFR ≤40 mL/min/1.73m2, shortened one-compartment models, in particular the 5-hour model, are less performant than the reference 8-hour model. For accurate GFR measurements, the iohexol plasma clearance should be measured with appropriate protocols. Over-simplified procedures should be avoided.

Background Tolvaptan and octreotide–long-acting release (LAR) have renoprotective effects in autosomal dominant polycystic kidney disease (ADPKD) that are partially mediated by amelioration of compensatory glomerular hyperfiltration. We compared the effects of tolvaptan and octreotide-LAR combination therapy versus those of tolvaptan monotherapy in patients with ADPKD. Methods This pilot, randomized, placebo-controlled, cross-over trial primarily compared the effects of 1- and 4-week treatments with octreotide-LAR (two 20-mg i.m. injections) or placebo (two i.m. 0.9% saline solution injections) added-on tolvaptan (up to 90 and 30 mg/d) on GFR (iohexol plasma clearance) in 19 consenting patients with ADPKD referred to a clinical research center in Italy. Analyses were intention-to-treat. The local ethical committee approved the study. Results At 4 weeks, GFR significantly decreased by a median (interquartile range) of 3 (−1 to 5) ml/min per 1.73 m 2 with tolvaptan and placebo ( P =0.01) and by 7 (3–14) ml/min per 1.73 m 2 with tolvaptan and octreotide-LAR ( P =0.03). GFR changes during the two treatment periods differed by 2 (−5 to 14) ml/min per 1.73 m 2 ( P =0.28). At 1 week, GFR significantly decreased by 3 (0–7) ml/min per 1.73 m 2 with tolvaptan and placebo ( P =0.006) and by 10 (−6 to 16) ml/min per 1.73 m 2 with tolvaptan and octreotide-LAR add-on therapy ( P <0.001). GFR changes during the two treatment periods significantly differed by 3 (0–12) ml/min per 1.73 m 2 ( P =0.012). Total kidney volume nonsignificantly changed by 4 (−48 to 23) ml with tolvaptan and placebo ( P =0.74), whereas it decreased significantly by 41 (25–77) ml with tolvaptan and octreotide-LAR ( P =0.001). Changes during the two treatment periods differed by 36 (0–65) ml ( P =0.01). Octreotide-LAR also attenuated ( P =0.02) the aquaretic effect of tolvaptan. Treatments were well tolerated. Conclusions In patients with ADPKD, octreotide-LAR added-on tolvaptan reduced GFR more effectively than octreotide-LAR and placebo. Octreotide-LAR also reduced total and cystic kidney volumes and attenuated the acquaretic effect of tolvaptan. Clinical Trial registry name and registration number: Tolvaptan-Octreotide LAR Combination in ADPKD (TOOL), NCT03541447.

Abstract BACKGROUND AND AIMS Neutrophils (Ns) play a key role in inflammation and tissue injury, in particular in acute renal failure, by releasing a complex of cytotoxic products such as reactive oxygen intermediates, enzymes and microbicidal polypeptides. These effects are prevented or mitigated by Ns depletion [1, 2]. Here we investigated the relationships between Ns count and the severity of kidney dysfunction in patients with chronic kidney disease (CKD). METHOD In this cross-sectional study we evaluated the association between circulating Ns absolute number, CKD stage and measured glomerular filtration rate (mGFR) in 393 patients with renal disease from different causes. CKD stage was established on the basis of mGFR and K/DOQI guidelines [3]. The relationships of Ns count and other considered covariates with mGFR were evaluated by uni- and multi-variable linear regression models (Table 1). Variables considered in univariable models are listed in Table 1. Variables included in multivariable models were identified with forward selection. Circulating Ns were measured by Coulter LH 780 Hematology Analyzer (Beckman Coulter, Milan, Italy). The GFR was measured by the iohexol plasma clearance technique, a gold standard for direct GFR measurement [4]. P < .05 was considered as statistically significant. RESULTS The mean ± SD age and mGFR of the patients were 54.64 ± 13.75 years and 62.75 ± 22.52 mL/min/1.73 m2 (range: 15.53–131.79 mL/min/1.73 m2), respectively. Most patients had a kidney function in stage 2 and 3a (Figure 1A). The absolute number of Ns averaged 4.32 ± 1.77 × 103µL−1. Ns counts increased progressively in parallel with increasing CKD stage (P < .005, Figure 1A) and were negatively correlated with mGFR (P < .001, Figure 1B). At multivariable analysis, mGFR was associated negatively with Ns count and positively with basophils (Bs) count (Table 1). CONCLUSION In patients with CKD higher numbers of Ns and lower numbers of Bs, even within the normal range, were independently associated with more severe renal insufficiency. A major limitation of the study is the cross-sectional design that does not allow to establish a causal relationship between Ns count and severity of kidney dysfunction. These data provide the basis for studies to investigate mechanisms explaining these associations.

Anemia can contribute to chronic allograft injury by limiting oxygen delivery to tissues, particularly in the tubulointerstitium. To determine mechanisms by which erythropoietin (EPO) prevents chronic allograft injury we utilized a rat model of full MHC-mismatched kidney transplantation (Wistar Furth donor and Lewis recipients) with removal of the native kidneys. EPO treatment entirely corrected post-transplant anemia. Control rats developed progressive proteinuria and graft dysfunction, tubulointerstitial damage, inflammatory cell infiltration, and glomerulosclerosis, all prevented by EPO. Normalization of post-transplant hemoglobin levels by blood transfusions, however, had no impact on chronic allograft injury, indicating that EPO-mediated graft protection went beyond the correction of anemia. Compared to syngeneic grafts, control allografts had loss of peritubular capillaries, higher tubular apoptosis, tubular and glomerular oxidative injury, and reduced expression of podocyte nephrin; all prevented by EPO treatment. The effects of EPO were associated with preservation of intragraft expression of angiogenic factors, upregulation of the anti-apoptotic factor p-Akt in tubuli, and increased expression of Bcl-2. Inhibition of p-Akt by Wortmannin partially antagonized the effect of EPO on allograft injury and tubular apoptosis, and prevented EPO-induced Bcl-2 upregulation. Thus non-erythropoietic derivatives of EPO may be useful to prevent chronic renal allograft injury.

Tolerance induction toward allogeneic organ grafts represents one of the major aims of transplantation medicine. Stem cells are promising candidates for promoting donor-specific tolerance. In this study, we investigated the immunomodulatory properties of murine embryonic stem cells (ESCs), obtained either by in vitro fertilization (IVF-ESCs) or by nuclear transfer (NT-ESCs), in heart transplant mouse models. IVF-ESCs did not prolong the survival of fully allogeneic cardiac transplants but significantly prolonged the survival of semiallogeneic hearts from the same ESC donor strain for >100 d in 44% of the animals. However, 28% of transplanted animals infused with IVF-ESCs experienced development of a teratoma. NT-ESCs similarly prolonged semiallogeneic heart graft survival (>100 d in 40% of the animals) but were less teratogenic. By in vitro studies, IVF-ESC and NT-ESC immunoregulation was mediated both by cell contact-dependent mechanisms and by the release of soluble factors. By adding specific inhibitors, we identified PGE2 as a soluble mediator of ESC immunoregulation. Expansion of regulatory T cells was found in lymphoid organs and in the grafts of IVF-ESC– and NT-ESC–tolerized mice. Our study demonstrates that both IVF-ESCs and NT-ESCs modulate recipient immune response toward tolerance to solid organ transplantation, and that NT-ESCs exhibit a lower tendency for teratoma formation. Because NT-ESCs are obtained by NT of a somatic cell from living individuals into an enucleated oocyte, they could represent a source of donor-derived stem cells to induce tolerance to solid organ allograft.

Background. Ischemia/reperfusion (I/R) injury is an important cause of renal graft dysfunction. Increases in cold and warm ischemia times lead to a higher risk of early posttransplant complications including delayed graft function and acute rejection. Moreover, prolonged cold ischemia is a predictor of long-term graft loss in kidney transplant patients. Methods. Darbepoetin alfa (DA) and carbamylated nonerythropoietic derivative of erythropoietin (CEPO) protective effects were evaluated in a model of I/R injury after kidney transplantation in both syngeneic and allogeneic combinations. The effects of wortmannin (phosphorylated Akt [p-Akt] inhibitor) administration were also investigated. Serum creatinine was evaluated at 16, 24, 48 hr and at 4 and 7 days posttransplant. Animals were killed 24 hr or 7 days after transplant and kidneys were processed for histological analysis, immunohistochemistry assessment of erythropoietin receptor (EPOR) and β-common chain receptor expression, granulocyte infiltration, nitrotyrosine staining, p-Akt expression, peritubular capillary (PTC) density, apoptosis, antioxidant, and antiapoptotic gene expression. Results. DA and CEPO significantly reduced serum creatinine, tubular injury, tubular nitrotyrosine staining, and prevented I/R-induced tubular apoptosis, but only when given both to the donor and to the recipient. DA and CEPO cytoprotection was associated with prevention of I/R-induced drop of p-Akt expression in tubuli, and almost complete preservation of capillary density in the tubulointerstitium of the graft. CEPO was more effective than DA in reducing tubular oxidative stress and preserving PTCs. Conclusion. DA and CEPO when given both to the donor and to the recipient, prevented renal graft dysfunction, tubular oxidative stress, and apoptosis after I/R injury in kidney transplantation. Their cytoprotection was mediated by tubular p-Akt activation and PTC density preservation.

Operational tolerance is a rare and still unexplained phenomenon in organ transplantation. Pallier et al. analyzed the peripheral B-cell compartment of 12 renal transplant patients with drug-free long-term graft function and found a peculiar blood B-cell phenotype, with an expansion of memory activated B cells and increased expression of inhibitory molecules, suggesting a role of B cells in maintaining graft tolerance. Kidney International (2010) 78, 435-437. doi:10.1038/ki.2010.159

Operational tolerance is a rare and still unexplained phenomenon in organ transplantation. Pallier et al. analyzed the peripheral B-cell compartment of 12 renal transplant patients with drug-free long-term graft function and found a peculiar blood B-cell phenotype, with an expansion of memory activated B cells and increased expression of inhibitory molecules, suggesting a role of B cells in maintaining graft tolerance. Kidney International (2010) 78, 435-437. doi:10.1038/ki.2010.159

Members of the TLR/IL-1R superfamily mediate ischemia/reperfusion injury and initiate immune response in transplanted organs. In this study, we tested the hypothesis that Toll-IL-1R8 (TIR8), a negative regulator of TLR/IL-1R highly expressed in the kidney, modulates immune cell activation underlying kidney rejection. In a mouse model of fully mismatched kidney allotransplantation in which the graft is spontaneously accepted, intragraft Tir8 expression was enhanced compared with naive kidneys. Targeted deletion of Tir8 in the graft exerted a powerful antitolerogenic action leading to acute rejection. Similarly, in a mouse model of kidney graft acceptance induced by costimulation blockade, most Tir8(-/-) grafts were acutely rejected. Despite similar levels of TLR4, IL-1R, and their ligands, the posttransplant ischemia/reperfusion-induced inflammatory response was more severe in Tir8(-/-) than in Tir8(+/+) grafts and was followed by expansion and maturation of resident dendritic cell precursors. In vitro, Tir8(-/-) dendritic cell precursors acquired higher allostimulatory activity and released more IL-6 upon stimulation with a TLR4 ligand and TNF-alpha than Tir8(+/+) cells, which may explain the increased frequency of antidonor-reactive T cells and the block of regulatory T cell formation in recipients of a Tir8(-/-) kidney. Thus, TIR8 acts locally as a key regulator of allogeneic immune response in the kidney. Tir8 expression and/or signaling in donor tissue are envisaged as a novel target for control of innate immunity and amelioration of graft survival.

In this study, we investigated whether mesenchymal stem cells (MSC) had immunomodulatory properties in solid organ allotransplantation, using a semiallogeneic heart transplant mouse model, and studied the mechanism(s) underlying MSC tolerogenic effects. Either single (portal vein, day −7) or double (portal vein, day −7 and tail vein, day −1) pretransplant infusions of donor-derived B6C3 MSC in B6 recipients induced a profound T cell hyporesponsiveness and prolonged B6C3 cardiac allograft survival. The protolerogenic effect was abrogated when donor-derived MSC were injected together with B6C3 hematopoietic stem cells (HSC), suggesting that HSC negatively impact MSC immunomodulatory properties. Both the induction (pretransplant) and the maintenance phase (>100 days posttransplant) of donor-derived MSC-induced tolerance were associated with CD4+CD25+Foxp3+ Treg expansion and impaired anti-donor Th1 activity. MSC-induced regulatory T cells (Treg) were donor-specific since adoptive transfer of splenocytes from tolerant mice prevented the rejection of fully MHC-mismatched donor-specific secondary allografts but not of third-party grafts. In addition, infusion of recipient-derived B6 MSC tolerized a semiallogeneic B6C3 cardiac allograft, but not a fully MHC-mismatched BALB/c graft, and expanded Treg. A double i.v. pretransplant infusion of recipient-derived MSC had the same tolerogenic effect as the combined intraportal/i.v. MSC infusions, which makes the tolerogenic protocol applicable in a clinical setting. In contrast, single MSC infusions given either peritransplant or 1 day after transplant were less effective. Altogether these findings indicate that MSC immunomodulatory properties require HSC removal, partial sharing of MHC Ags between the donor and the recipient and pretransplant infusion, and are associated with expansion of donor-specific Treg.

Ischemia-reperfusion injury is an important cause of graft failure. Because carnitine regulates substrate flux and energy balance across membranes which may be deranged in ischemia we determined whether its use was effective in preventing kidney injury in an allogeneic transplant model. Brown Norway rats received a Lewis rat kidney transplant and were then treated with cyclosporine A to avoid rejection. The grafts were stored in Belzer solution supplemented with propionyl-L-carnitine during the cold ischemia period. Compared to rats receiving untreated kidneys but with equal cold ischemia times, the post-transplant serum creatinine values of the carnitine-treated transplants were significantly lower. Histological evaluation 16 h after transplant showed that propionyl-L-carnitine significantly inhibited tubular necrosis and neutrophil infiltration of the allografts and improved the 3 month graft survival. Treated transplants also had decreased lipid peroxidation, inducible nitric oxide synthase expression and protein nitration compared to the untreated grafts. Post-transplant serum creatinine levels were significantly reduced and graft survival was slightly prolonged in rats not receiving cyclosporine A treatment and transplanted with a kidney treated with propionyl-L-carnitine. The efficacy of propionyl-L-carnitine to modulate ischemia-reperfusion injury during transplantation suggests that its use in human transplantation is worth testing.

Background. We previously documented that rat bone marrow-derived dendritic cells (DCs), transfected with an adenovirus encoding a dominant negative form of IKK2 (dnIKK2), have impaired allostimulatory capacity and generate CD4+ T cells with regulatory function. Here we investigate the potency, the phenotype, and the mechanism of action of dnIKK2-DC-induced regulatory cells and we evaluated their tolerogenic properties in vivo. Methods. Brown Norway (BN) transfected dnIKK2-DCs were cultured with Lewis (LW) lymphocytes in primary mixed lymphocyte reaction (MLR). CD4+ T cells were purified from primary MLR and incubated in secondary coculture MLR with LW lymphocytes. Phenotypic characterization was performed by fluorescence-activated cell sorting and real-time polymerase chain reaction. The tolerogenic potential of CD4+ T cells pre-exposed to dnIKK2-DCs was evaluated in vivo in a model of kidney allotransplantation. Results. CD4+ T cells pre-exposed to dnIKK2-DCs were CD4+CD25−/dim and expressed interleukin (IL)-10, transforming growth factor-beta, interferon-gamma, IL-2, and inducible nitric oxide synthase (iNOS). These cells (dnIKK2-Treg), cocultured (at up to 1:105 ratio) with a primary MLR, suppressed T-cell proliferation to alloantigens. The regulatory effect was cell-to-cell contact-independent since it was also observed in a transwell system. A nitric oxide synthase inhibitor significantly reverted dnIKK2-Treg-mediated suppression, whereas neutralizing antibodies to IL-10 and TGF-beta had no significant effect. DnIKK2-Treg given in vivo to LW rats prolonged the survival of a kidney allograft from BN rats (the donor rat strain used for generating DCs). Conclusions. DnIKK2-Treg is a unique population of CD4+CD25− T cells expressing high levels of iNOS. These cells potently inhibit T-cell response in vitro and induce prolongation of kidney allograft survival in vivo.

Background. In kidney transplant recipients with alemtuzumab induction maintained on mycophenolate mofetil (MMF) immunosuppression, sirolimus (SRL) promotes significant expansion of circulating CD4+CD25high regulatory T cells (Treg). This might translate into more effective protection against chronic graft injury compared to cyclosporin A (CsA), which, in the same clinical setting, does not affect Treg. Methods. To assess this hypothesis, in the extension of a single-center, prospective, randomized, open, blind endpoint study aimed to assess the effect of low-dose SRL or CsA on circulating Treg, we compared the outcomes of renal transplant recipients on SRL (n=11) or CsA (n=10) by per-protocol biopsies and serial measurements of glomerular filtration rate (GFR), renal plasma flow (RPF), and 24-hour proteinuria over 30 months posttransplant. Results. Despite 4-fold higher CD4+CD25high Treg counts (22.1±12.2% vs. 5.7±4.2% of CD3+CD4+ T cells), SRL-treated patients, compared to CsA-treated patients, had a significantly higher tubular C4d staining score (1.1±0.6 vs. 0.2±0.3, P<0.01), with nonsignificant trends to higher chronic allograft damage index score (5.6±2.4 vs. 3.7±3.3), faster GFR (−2.92±0.33 vs. −0.28±0.44 ml/min/1.73m2 per year), and RPF (−10.80±5.45 vs. −1.86±3.09 ml/min/1.73 m2 per year) decline, and more clinical proteinuria (n=6 vs. 4). There was no significant correlation between Treg counts and any considered outcome variable in the study group as a whole and within each cohort. Conclusions. These data suggest that, despite enhanced Treg expression, low-dose SRL combined to alemtuzumab induction and MMF-based steroid-free maintenance therapy, does not appreciably protect renal transplant recipients from chronic allograft injury and dysfunction.

Allogeneic immune responses are modulated by a subset of host T cells with regulatory function (Treg) contained within the CD4(+)CD25(high) subset. Evidence exists that Treg expand after peritransplantation lymphopenia, inhibit graft rejection, and induce and maintain tolerance. Little, however, is known about the role of Treg in the clinical setting. IL-2 and activation by T cell receptor engagement are instrumental to generate and maintain Treg, but the influence of immunosuppressants on Treg homeostasis in humans in vivo has not been investigated. This study monitored Treg phenotype and function during immune reconstitution in renal transplant recipients who underwent profound T cell depletion with Campath-1H and received sirolimus or cyclosporine (CsA) as part of their maintenance immunosuppressive therapy. CD4(+)CD25(high) cells that expressed FOXP3 underwent homeostatic peripheral expansion during immune reconstitution, more intense in patients who received sirolimus than in those who were given CsA. T cells that were isolated from peripheral blood long term after transplantation were hyporesponsive to alloantigens in both groups. In sirolimus- but not CsA-treated patients, hyporesponsiveness was reversed by Treg depletion. T cells from CsA-treated patients were anergic. Thus, lymphopenia and calcineurin-dependent signaling seem to be primary mediators of CD4(+)CD25(high) Treg expansion in renal transplant patients. These findings will be instrumental in developing "tolerance permissive" immunosuppressive regimens in the clinical setting.