Deceased donor procurement biopsy practices, interpretation, and histology-based decision-making: A survey of US kidney transplant centers

INTRODUCTION: The utility of kidney procurement biopsies is controversial. Understanding the current landscape of how clinicians obtain and use biopsies in organ evaluation may help inform consensus-building efforts. METHODS: An electronic survey was distributed to clinicians at US kidney transplant programs (April 22, 2021-June 30, 2021) to evaluate donor biopsy indications, frequency, processing and interpretation, and impact of findings on practices. RESULTS: Responses from staff involved in organ acceptance (73% surgeons, 20% nephrologists, 6% coordinators) at 95 transplant centers were analyzed, representing 40% of US transplant centers and 50% of recent deceased donor kidney transplant volume. More than a third of centers (35%) reported obtaining procurement biopsies on most-to-all kidneys. Most clinicians decided when to biopsy jointly with the Organ Procurement Organization (OPO) (82%) based on formal criteria for the decision (72%), although 41% reported having requested a biopsy outside of the criteria. Most respondents used a semiquantitative scoring system for interpretation (57%). Many respondents reported rarely or never having access to renal specialty pathologists (37%) or to telepathology (59%). Most respondents reported that a favorable biopsy result would encourage them to accept a marginal donor kidney (72%); nearly half (46%) indicated that an unfavorable biopsy result would lead to decline of a standard criteria kidney. CONCLUSION: Procurement biopsies are commonly used in organ acceptance decisions despite inconsistent access to experienced renal pathologists and heterogeneous approaches to criteria, scoring, and interpretation. Ongoing study and consensus building are needed to direct procurement biopsy practice toward increasing organ utilization and reducing allocation inefficiency

NAC and DTT promote TGF-β1 monomer formation: demonstration of competitive binding

TGF-β plays an important role in the genesis and progression of pulmonary fibrosis. We sought to determine the role of mononuclear phagocytes in the activation of TGF-β and found that freshly isolated peripheral blood monocytes spontaneously released TGF-β. Stimulating these monocytes with GM-CSF or LPS, but not MCSF, augmented the activation of TGF-β. In human monocytes, the free thiol compounds DTT and NAC decreased the activity of TGF-β, without affecting TGF-β mRNA transcription. Both NAC and DTT lessened the biological activity of recombinant active TGF-β in a cell-free system. We found that NAC and DTT reduced dimeric active TGF-β from a 25 kDa protein to 12.5 kDa inactive monomer. This conversion was reversed using the oxidizing agent diamide. Diamide also restored biological activity to NAC or DTT-treated TGF-β. Reduction of TGF-β to monomers could competitively inhibit active dimeric TGF-β and block intracellular signaling events. Our observations suggest that modulation of the oxidative state of TGF-β may be a novel therapeutic approach for patients with pulmonary fibrosis

The CAT-ACT Beamline at ANKA: A new high energy X-ray spectroscopy facility for CATalysis and ACTinide research

A new hard X-ray beamline for CATalysis and ACTinide research has been built at the synchrotron radiation facility ANKA. The beamline design is dedicated to X-ray spectroscopy, including ‘flux hungry’ photon-in/photon-out and correlative techniques with a special infrastructure for radionuclide and catalysis research. The CAT-ACT beamline will help serve the growing need for high flux/hard X-ray spectroscopy in these communities. The design, the first spectra and the current status of this project are reported

Latency Associated Peptide Has In Vitro and In Vivo Immune Effects Independent of TGF-β1

Latency Associated Peptide (LAP) binds TGF-β1, forming a latent complex. Currently, LAP is presumed to function only as a sequestering agent for active TGF-β1. Previous work shows that LAP can induce epithelial cell migration, but effects on leukocytes have not been reported. Because of the multiplicity of immunologic processes in which TGF-β1 plays a role, we hypothesized that LAP could function independently to modulate immune responses. In separate experiments we found that LAP promoted chemotaxis of human monocytes and blocked inflammation in vivo in a murine model of the delayed-type hypersensitivity response (DTHR). These effects did not involve TGF-β1 activity. Further studies revealed that disruption of specific LAP-thrombospondin-1 (TSP-1) interactions prevented LAP-induced responses. The effect of LAP on DTH inhibition depended on IL-10. These data support a novel role for LAP in regulating monocyte trafficking and immune modulation

Critical roles of calpastatin in ischemia/reperfusion injury in aged livers

Ischemia/reperfusion (I/R) injury unavoidably occurs during hepatic resection and transplantation. Aged livers poorly tolerate I/R during surgical treatment. Although livers have a powerful endogenous inhibitor of calpains, calpastatin (CAST), I/R activates calpains, leading to impaired autophagy, mitochondrial dysfunction, and hepatocyte death. It is unknown how I/R in aged livers affects CAST. Human and mouse liver biopsies at different ages were collected during in vivo I/R. Hepatocytes were isolated from 3-month- (young) and 26-month-old (aged) mice, and challenged with short in vitro simulated I/R. Cell death, protein expression, autophagy, and mitochondrial permeability transition (MPT) between the two age groups were compared. Adenoviral vector was used to overexpress CAST. Significant cell death was observed only in reperfused aged hepatocytes. Before the commencement of ischemia, CAST expression in aged human and mouse livers and mouse hepatocytes was markedly greater than that in young counterparts. However, reperfusion substantially decreased CAST in aged human and mouse livers. In hepatocytes, reperfusion rapidly depleted aged cells of CAST, cleaved autophagy-related protein 5 (ATG5), and induced defective autophagy and MPT onset, all of which were blocked by CAST overexpression. Furthermore, mitochondrial morphology was shifted toward an elongated shape with CAST overexpression. In conclusion, CAST in aged livers is intrinsically short-lived and lost after short I/R. CAST depletion contributes to age-dependent liver injury after I/R

LAP-induced monocyte chemotaxis is blocked by specific inhibitors of LAP-TSP-1 interactions.

<p>Human monocytes (5×10⁴/condition) were suspended in Gey's balanced salt solution and assayed for their chemotactic response to rhLAP. Recruitment was assayed by incubating monocytes in modified Boyden chambers for 90 minutes at 37°C and quantified by counting five high-powered fields of stained membranes in a blinded fashion and reported as fold-increase of LAP (10 pg/ml)-induced monocyte chemotaxis compared to monocyte chemotaxis induced by media alone. rhLAP at 10 pg/ml was chosen based on previous experiments that determined this dose induced maximal cellular recruitment. These data represent n = 3 (minimum) separate experiments expressed as the mean fold-change±SEM. Reported p values compare fold-change in monocyte chemotaxis induced by LAP without and with identified inhibitors.</p

Latency-associated peptide (LAP) induces monocyte chemotaxis via a pathway distinct from TGF-β1.

<p>C57BL/6 female mice were anesthetized and subcutaneously injected with Matrigel™ matrix supplemented with PBS, rhLAP or CCL2/MCP-1. After 10 days the mice were sacrificed, skinned, and the plugs were removed, fixed and embedded in paraffin then stained for CD68+ cells (mononuclear phagocytes). Matrigel matrix supplemented with (A) PBS, (B) 10 pg/ml rhLAP, or (C) 10 ng/ml CCL2 were assessed for CD68+ cell recruitment and then quantified by blinded counts (D). Arrow heads indicate CD68+ cells. (*p<0.05 for condition vs. PBS control plugs, n = 3 for CCL2 and n = 2 for LAP) Human monocytes (5×10⁴/condition) were suspended in Gey's balanced salt solution and assayed for their chemotactic response to rhLAP. Recruitment was assayed by incubating monocytes in modified Boyden chambers for 90 minutes at 37°C and quantified by counting five high-powered fields of stained membranes in a blinded fashion. (E) Increasing doses of rhLAP (0-100 pg/ml) were used to stimulate monocyte recruitment (†p<0.001 from the non-stimulated sample, n = 7). (F) Analysis for evidence of directed recruitment of monocytes by LAP was assessed by exposing monocytes to a “true” gradient of rhLAP (10 pg/ml) (rhLAP in the chamber opposite the monocytes <i>only</i>; dark bars–“Monocytes”) or no gradient (rhLAP on both sides of the chamber; lighter bars–“Monocytes+LAP”). These data represent two independent studies done in triplicate (*p<0.001 vs. mono + LAP condition). (G) Assessment of LAP-specific chemotaxis was performed by pre-incubating cell suspensions with the SB 431542 hydrate from Sigma (St.Louis, MO), a selective inhibitor of TGF-β type 1 receptor kinases (Activin Receptor-Like Kinases, ALK-4,-5, and-7) 20 minutes prior to loading on chemotaxis chambers. Monocytes were then exposed to optimal chemotactic doses of LAP (10 pg/ml) and TGF-β1 (100 pg/ml). All bars represent the mean±SEM for n = 4 samples (* p<0.05 by ANOVA with post-hoc testing).</p

LAP is able to suppress <i>in vivo</i> cellular inflammation similar to TGF-β1 through interactions with thrombospondin-1.

<p>A transfer DTHR assay was performed with antigen (tetanus toxin) and tested agents (TGF-β1, rhLAP and anti-TGF-β1, LAP or TSP-1). C57BL/6 mice were sensitized to tetanus toxin (TT) and then tested to confirm they had developed a TT-induced DTHR. DTHR was induced in the pinnae of naïve C57BL/6 mice with simultaneous injection (35 µl) of antigen and splenocytes (8×10⁶ cells/condition) harvested from antigen-sensitized mice. DTHR was quantified by measurement of ear thickness 24 hours after injection. (A) LAP (10 pg) and TGF-β1 (5 ng) were tested for the ability to reduce DTHR when used alone, with anti-TGF-β1 antibodies (1 µg) or anti-LAP antibodies (1 µg) (*p<0.02 versus tetanus sensitized splenocytes + TT; †p<0.01 versus tetanus sensitized splenocytes + TT, n = 5). Similar results were observed when this assay was repeated using C57BL/6 mice that had rejected a cardiac allograft (DBA/2->C57BL/6) as a model of transfer DTHR. (B) Anti-TSP-1 antibody or isogenic control IgG (all at 1 µg) were tested for their ability to interfere with rhLAP suppression of TT-induced DTHR (*p<0.02 versus tetanus sensitized splenocytes + TT; †p<0.01 versus tetanus sensitized splenocytes + TT, n = 3). (C) Transfer DTHR assay was performed with antigen, rhLAP and the inhibitory peptide, LSKL or its scrambled control SLLK (20 µM) (†p<0.01 versus tetanus-sensitized splenocytes + TT, n = 2). (D) To determine if LAP used an IL-10 dependent pathway to inhibit the DTHR (as previously published) the DTHR transfer assay was repeated with anti-IL-10 antibodies and isotype control. (1 µg each) (*p<0.01 vs. tetanus-sensitized splenocytes + TT, n = 3) All bars represent the mean±SEM. DTHR, delayed-type hypersensitivity response; Ag, Antigen; BKG, background ear girth.</p

LAP-induced inflammatory cell recruitment is altered in TSP-1 deficient (TSP-1 −/−) mice.

<p>(A) Bone marrow macrophages from wild type C57BL/6 or TSP-1−/− mice were isolated and assessed for chemotaxis activity using modified Boyden chambers. Macrophages (5×10⁴/condition) were suspended serum-free in Gey's balanced salt solution and chemotaxis was assayed by counting the mean of five blinded high-powered fields on the polycarbonate filter (†p<0.001 for the chemotaxis of wild-type vs. TSP-1 −/− BMM). The data is expressed as fold-change in the mean compared to control (media alone)±SEM for two independent studies done in triplicate. (B) LAP's ability to affect the direct DTHR was tested in TSP-1 −/− and wild-type mice. TSP-1 −/− and wild-type C57BL/6 mice were sensitized to tetanus toxin (TT) and then tested to confirm they had developed a TT-induced DTHR. This direct DTHR assay was performed in the pinnae of these mice with simultaneous injection (35 µl) of antigen (tetanus toxin) alone or with rhLAP (10 pg) (†p = 0.004 for LAP-treated versus untreated WT mice, n = 3). All bars represent the mean±SEM for two studies unless otherwise noted. DTHR, delayed-type hypersensitivity response; TT, tetanus toxin; WT, wild type, BKG, background ear girth.</p