The KTx360°-study: a multicenter, multisectoral, multimodal, telemedicine-based follow-up care model to improve care and reduce health-care costs after kidney transplantation in children and adults.

BACKGROUND: Follow-up care after kidney transplantation is performed in transplant centers as well as in local nephrologist's practices in Germany. However, organized integrated care of these different sectors of the German health care system is missing. This organizational deficit as well as non-adherence of kidney recipients and longterm cardiovascular complications are major reasons for an impaired patient and graft survival. METHODS: The KTx360° study is supported by a grant from the Federal Joint Committee of the Federal Republic of Germany. The study will include 448 (39 children) incident patients of all ages with KTx after study start in May 2017 and 963 (83 children) prevalent patients with KTx between 2010 and 2016. The collaboration between transplant centers and nephrologists in private local practices will be supported by internet-based case-files and scheduled virtual visits (patient consultation via video conferencing). At specified points of the care process patients will receive cardiovascular and adherence assessments and respective interventions. Care will be coordinated by an additional case management. The goals of the study will be evaluated by an independent institute using claims data from the statutory health insurances and data collected from patients and their caregivers during study participation. To model longitudinal changes after transplantation and differences in changes and levels of immunosuppresive therapy after transplantation between study participants and historical data as well as data from control patients who do not participate in KTx360°, adjusted regression analyses, such as mixed models with repeated measures, will be used. Relevant confounders will be controlled in all analyses. DISCUSSION: The study aims to prolong patient and graft survival, to reduce avoidable hospitalizations, co-morbidities and health care costs, and to enhance quality of life of patients after kidney transplantation. TRIAL REGISTRATION: ISRCTN29416382 (retrospectively registered on 05.05.2017).

[Rehabilitation after kidney transplantation: Old problems and new structures]. / Rehabilitation nach Nierentransplantation: Alte Probleme und neue Strukturen.

Kidney transplantation is currently the best therapeutic option for patients with end stage renal disease. Alternative treatment with hemo- or peritoneal dialysis is associated with higher comorbidities, higher morbidity/mortality, and reduced quality of life. Thus, a major aim in posttransplant care is to develop strategies to increase transplant survival and reduce known risk factors and comorbidities. In this overview, we propose a concept to include rehabilitation clinics in all aspects of the transplant process. This concept includes pretransplant care on the waiting list to prepare the patient for the transplant, the direct postoperative treatment phase, and repeated and risk adapted stays in rehabilitation clinics during long-term follow-up to address specific and individual problems.

CXCL13 as a new biomarker of systemic lupus erythematosus and lupus nephritis - from bench to bedside?

Different studies over the last decade have linked the B cell-attracting chemokine CXC ligand 13 (CXCL13) to the autoimmune disease systemic lupus erythematosus (SLE). A pathogenetic role of this chemokine for disease manifestation in SLE was described initially in mouse models for SLE. Mechanisms of CXCL13 actions were also identified in SLE patients. Moreover, various clinical studies have identified CXCL13 serum levels as a useful biomarker in patients with SLE of different ethnicities for disease activity. In addition, CXCL13 seems to be a promising marker for the diagnosis of lupus nephritis, one of the most severe complications of SLE. However, its exact place within the mechanisms that lead to SLE remains to be defined. Further research is needed to resolve more details of the pathomechanism and the signalling pathway of CXCL13 in SLE. Blocking CXCL13 or the signal pathways of CXCL13 is seen as a promising therapeutic approach for SLE and will be addressed in the near future. This review summarizes all papers that linked CXCL13 to SLE and highlights its importance in the pathogenesis and diagnosis of SLE.

Pathogenetic role of glomerular CXCL13 expression in lupus nephritis.

Podocytes maintain the structure and function of the glomerular filtration barrier. However, podocytes have recently been implicated in the innate immune response, and their function as non-haematopoietic antigen-presenting cells was highlighted. We have shown previously that excessive expression of the chemokine CXCL13 is a distinctive early event for nephritis in a murine model of systemic lupus erythematosus (SLE). Furthermore, we found that CXCL13 is elevated significantly in the serum of patients with SLE-nephritis. In this study, we were able to show for the first time that (i) CXCL13 is expressed locally in glomeruli in a model for SLE-nephritis in mice and that (ii) incubation of human podocytes with CXCL13 induces receptor stimulation of CXCR5 with activation of signalling pathways, resulting in (iii) secretion of proinflammatory cytokines and chemokines in culture supernatant. This cytokine/chemokine cocktail can lead to (iv) a neutrophil respiratory burst in isolated human granulocytes. Taken together, our results provide further evidence that CXCL13 is involved in the pathogenesis of glomerulonephritis and that podocytes can play an active role in local proinflammatory immune responses. Thus, CXCL13 could be a direct target for the therapy of glomerulonephritis in general and for SLE-nephritis in particular.

CXCL13 as a novel marker for diagnosis and disease monitoring in pediatric PTLD.

Posttransplant lymphoproliferative disease (PTLD) is a severe complication of immunosuppressive treatment in organ-grafted children. Early diagnosis of PTLD is hampered by both unspecific clinical symptoms and lack of easy accessible markers. The homeostatic chemokine CXCL13, which plays a crucial role in B-cell homing and lymphoid organ development, is expressed in some lymphomatous diseases. This study aims to investigate whether serum CXCL13 (sCXCL13) levels correlate with occurrence and regression of PTLD in pediatric solid-organ graft recipients. Serum samples from PTLD patients (n = 21), patients with Epstein-Barr virus (EBV) reactivation (n = 18), and healthy age-matched controls (n = 19) were tested for CXCL13 using a commercially available ELISA kit. sCXCL13 levels were significantly higher in PTLD patients than in healthy children. PTLD patients had also higher sCXCL13 values than pediatric solid-organ recipients with EBV reactivation. An increase in sCXCL13 levels was observed from EBV reactivation to PTLD diagnosis in most cases. Elevated sCXCL13 levels were detected up to 2 years prior to PTLD diagnosis and correlated well with response to cytoreductive treatment in individual patients. sCXCL13, thus, may be a readily available surrogate marker for the diagnosis of PTLD and for monitoring of response to treatment in patients with initially elevated sCXCL13 levels.

Elevation of serum CXCL13 in SLE as well as in sepsis.

Recent studies have demonstrated that CXCL13 serum levels correlate significantly with systemic lupus erythematosus (SLE) disease activity. However, experimental studies show that CXCL13 production can also be induced by bacterial exposure as well as in response to inflammatory cytokines. This report asks whether CXCL13 serum levels are elevated in patients with evidence of bacterial infections and whether there is a correlation with the C-reactive protein (CRP) levels or the severity of illness in critically ill patients. CXCL13 levels were compared in 39 patients with active SLE (without concomitant infection), 40 non-SLE patients with sepsis, and 40 healthy controls by enzyme-linked immunosorbent assay (ELISA) methodology. We also tested storage conditions and freeze-thaw cycles for stability of CXCL13 in serum samples. Our studies demonstrated that the median CXCL13 serum levels were significantly elevated in patients with SLE [median 83 pg/ml (interquartile range 38-366)] or sepsis [359 pg/ml (151-459)] compared with healthy controls [32 pg/ml (27-41), p < 0.001]. The CXCL13 serum levels correlated with disease activity in SLE (CXCL13 vs. SLEDAI r = 0.65, p < 0.001), but were not associated with severity of illness score in critically ill patients (CXCL13 vs. SOFA r = -0.15, p = 0.35). However, CXCL13 serum levels were clearly associated with CRP levels in both sepsis (r = 0.45, p = 0.003) and SLE (r = 0.39, p = 0.02). In conclusion, CXCL13 is a stable serum marker for disease activity in SLE patients, but concomitant infections can also lead to increased CXCL13 levels.

Erythropoietic and reticuloendothelial function in bone marrow in dogs.

Erythropoietic and reticuloendothelial functions in bone marrow were found to be identically distributed between various bones and within individual bones in the dog.

FIT & STRONG! PLUS: DESCRIPTIVE DEMOGRAPHIC AND RISK CHARACTERISTICS IN A COMPARATIVE EFFECTIVENESS TRIAL FOR OLDER AFRICAN-AMERICAN ADULTS WITH OSTEOARTHRITIS.

OBJECTIVES: The prevalence of osteoarthritis (OA) has increased in the US. We report on a comparative effectiveness trial that compares Fit & Strong!, an existing evidence-based physical activity (PA) program, to Fit & Strong! Plus, which combines the Fit & Strong! intervention with a weight management intervention. METHODS: Participants included 413 overweight/obese (BMI 25-50 kg/m2) adults with lower extremity (LE) OA. The majority of the sample was African-American and female. Both interventions met 3 times weekly for 8 weeks. Primary measures included diet and weight. RESULTS: The baseline mean BMI for all participants was 34.8 kg/m², percentage of calories from fat was high, and self-reported PA was low. DISCUSSION: This sample of overweight/obese African-American adults had lifestyle patterns at baseline that were less than healthful, and there were differences between self-report and performance-based measures as a function of age.

The steroid metabolite 16(ß)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1.

The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(ß)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased Km and decreased kcat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites.

Growth kinetics of mammary tumor 13762 in rats previously cured by chemotherapy.

We studied the growth capabilities of mammary tumor 13762 transplanted into inbred F344 rats previously cured of tumors by cell kinetically based sequential chemotherapy. Of the 18 challenge tumors, 4 were completely rejected, and nonrejected tumors grew at subnormal rates. The subnormal growth was specific for the cured rats because tumor growth in age- and therapy-matched non-tumor-bearing controls was normal. Cell kinetic studies with the use of in vitro techniques for the [3H]dThd labeling index, DNA synthesis time, and primer-dependent DNA polymerase labeling index (an in vitro estimate of growth fraction) indicated that the subnormal growth rates of the 13762 tumor in cured rats were due to subnormal tumor cell production. Cell loss rates were similar in tumors growing in cured rats and in size-matched tumors growing in normal controls. The results are consistent with the possibility that the subnormal growth of 13762 challenge tumors in chemotherapeutically cured F344 rats was mediated by immune factors.

Cell kinetics in vivo and in vitro for C3H/He spontaneous mammary tumors.

Cell kinetics in spontaneous C3H/HeJ mammary tumors of retired-breeder mice was studied by in vivo and in vitro techniques. The [3H]TdR labeling index (LI), the DNA synthesis time (TS), and the primer-dependent DNA polymerase assay LI [an in vitro estimate of tumor growth fraction (GF)] were compared to similar measurements made in vivo. These measurements as well as the calculated cell kinetic parameters derived from these data were not different in in vivo and in vitro studies. Furthermore, the cell kinetic parameters in tumors classified histologically as type A or type B mammary tumors were also similar. Although considerable variation in volume doubling times (Td's), [3H]TdR LI's, potential doubling times, cell cycle times (Tc's), and cell loss was found, Ts's were similar in all mammary tumors. No correlation between tumor volume or tumor weight and cell kinetic parameters was seen. However, the most slowly growing tumors (i.e., tumors with the longest Td's) tended to have the lowest [3H]TdR LI's, the longest Tc's, and the highest cell loss factors. No correlation was found between the GF and Td. However, tumors with the most rapidly proliferating cell populations tended to have the highest GF's.

Effect of methylprednisolone on the cell kinetic response of C3H/HeJ mammary tumors to cyclophosphamide and adriamycin.

The effect of methylprednisolone (MP) on the cell kinetic response to cyclophosphamide (CP) and Adriamycin (ADR) in C3H/HeJ spontaneous mammary tumors and hematopoietic tissue was investigated. The [3H]deoxythymidine labelingg index, the primer-dependent DNA polymerase labeling index (an estimate of tumor growth fraction), and the mitotic index were determined at various intervals after treatment. Treatment consisted of CP (200 mg/kg) on Day 0 plus ADR (2 mg/kg) on Day 1 with or without MP every 12 hr for 9 doses beginning on Day 2. In tumors treated with CP and ADR alone, changes in the kinetic parameters suggested proliferative recovery between Days 3 and 4 which coincided with bone marrow recovery. In tumors treated with CP, ADR, and MP, although the timing of the hematopoietic recovery was not affected by MP, the overshoot of the [3H]deoxythymidine labelin index on Days 3 and 4 was abolished. Proliferative recovery in the tumor was delayed until after cessation of MP treatments. Cell kinetic changes in the tumor after CP, ADR, and MP were used to design effective sequential chemotherapy which obviated the hematopoietic toxicity associated with sequential therapy designed from cell kinetic changes after CP and ADR alone.

Cell kinetic-directed sequential chemotherapy with cyclophosphamide and adriamycin in T1699 mammary tumors.

The present studies were initiated to investigate the changes [3H]deoxythymidine labeling index, primer-dependent DNA polymerase labeling index, and S-G2 transition after treatment of T1699 transplantable mouse mammary tumors with Adriamycin (5 mg/kg) and cyclophosphamide (100 mg/kg). Treatment with these agents resulted in intervals of subnormal tumor cell proliferation as indicated by decreased [3H]deoxythymidine labeling index, primer-dependent DNA polymerase labeling index, and S-G2 transition. Recovery, as indicated by increases in [3H]deoxythymidine labeling index, primer-dependent DNA polymerase labeling index, and S-G2 transition, was observed three days after Adriamycin treatment and six to seven days after cyclophosphamide treatment. To evaluate the predictive nature of the kinetic changes for effective time sequencing, sequential combination chemotherapy protocols were designed and tested in T1699 tumor-bearing mice. The results from these studies showed that the most effective chemotherapy schedules were those in which the drugs were sequenced to coincide with the cell kinetic recovery from the single agents alone. These effective sequencing intervals were also found to be effective when used in multifraction sequential combination chemotherapy protocols. The results suggest that changes in cell kinetic parameters following drug perturbation can provide indications as to potentially efficacious as well as nonefficacious sequencing intervals.

Antiproliferative effects of corticosteroids in C3H/HeJ Mammary tumors and implications for sequential combination chemotherapy.

Corticosteroid-induced inhibition of cell proliferation and tumor growth was studied in first-generation transplants (FGMT) of spontaneous C3H/HeJ mammary tumors (SMT). Competitive binding studies using the dextran-coated charcoal method demonstrated that both SMT and FGMT exhibit high-affinity, low-capacity cytoplasmic binding sites for corticosteroids. Free cytoplasmic binding sites, determined by Scatchard analysis of dexamethasone (DEX) binding data, were more abundant in SMT (323 +/- 45 fmol/mg) than in FGMT (199 +/- 35 fmol/mg). Apparent dissociation constants, 3.83 +/- 1.14 and 5.06 +/- 1.53 nM for SMT and FGMT, respectively, were consistent with those found in other corticosteroid-sensitive tissues. In vivo treatment of FGMT with DEX or methylprednisolone resulted in dose-dependent inhibition of cell proliferation and tumor growth. The recovery kinetics after three doses of either methylprednisolone or DEX (10 mg/kg every 12 hr) suggested reversible G1 progression delay. Changes in the [3H]thymidine labeling index after steroid treatment indicated maximal S-phase cellularity 18 to 24 hr after methylprednisolone and 42 to 48 hr after DEX. On the basis of regrowth delay measurements, the effectiveness of sequential therapy with corticosteroids and either 5-fluorouracil or especially vincristine was seen to be time sequence dependent. The most effective intervals were those in which vincristine and/or 5-fluorouracil was given to coincide with maximal S-phase cellularity after steroid treatments.

Effect of dexamethasone on vascular function in RIF-1 tumors.

The present series of experiments was conducted to determine the effect of dexamethasone on vascular function and cell proliferation in s.c. RIF-1 tumors. 125I-BSA, 51Cr-EDTA dilution techniques were used to evaluate dexamethasone induced changes in tumor plasma water, capillary permeability, and extracellular water volumes, while 59Fe and 51Cr labeled erythrocyte techniques were used to assess changes in tumor exchangeable erythrocyte volumes. 86RbCl distribution studies were also conducted to evaluate vascular perfusion in RIF-1 tumors after dexamethasone treatment. In this corticosteroid receptor containing tumor model, dexamethasone had profound effects on all of the measured parameters of vascular function. Reduced tumor cell proliferation after dexamethasone treatments was accompanied by reduced capillary permeability, reduced interstitial water volumes, increased plasma volumes, and reduced vascular perfusion. Serial studies after dexamethasone treatments indicated that increases in vascular perfusion preceded proliferative recovery. Intervals of maximal [3H]thymidine labeling after dexamethasone were characterized by transient increases in capillary permeability, interstitial water volumes, and tumor erythrocyte exchange with the general circulation. At intervals of maximal cell proliferation (36-48 h after dex) 86RbCl distribution in tumors was about 3 times that seen in untreated controls. The results seem to indicate that, as in edematous normal tissues, dexamethasone can have profound effects on vascular function and water compartmentalization in RIF-1 tumors.

Adriamycin-induced delayed erythropoietic injury expressed following anemia stress.

The present studies were undertaken to compare anemia-induced erythropoietic responses in femoral marrows and spleens of mice pretreated with Adriamycin (ADR) or 1-beta-D-arabinofuranosylcytosine with those of untreated age-matched controls. Mice were bled 45 or 120 days after drug treatment. The erythropoietic response to bleeding was quantitated by morphological, gravimetric, and radioiron methods 48 hr after bleeding. At 120 days after ADR, prebleeding base-line cellularity parameters were, in general, similar to those found in untreated age-matched controls. The response to the anemia stress was compared in drug-treated animals and in age-matched untreated controls, and the response deficit was expressed as residual injury (RI). At 120 days, ADR-induced RI was observed to be dose dependent in both femoral marrow and spleen. ADR-induced RI in femoral marrow and spleen were similar at 45 and 120 days, with no significant recovery. Although marrow RI was noted 45 days after 200 mg 1-beta-D-arabinofuranosylcytosine per kg, there was no RI at 120 days. The results indicate that ADR can induce a long-lasting hematopoietic injury which is not obvious from measures of homeostatic cellularity, but which can be expressed after induction of an acute proliferative demand.

Receptor-dependent antiproliferative effects of corticosteroids in radiation-induced fibrosarcomas and implications for sequential therapy.

Competitive binding studies with [3H]dexamethasone and Scatchard analysis demonstrated a single class of high-affinity, low-capacity glucocorticoid receptor sites in 105,000 x g cytosols from radiation-induced fibrosarcomas. In vivo, both dexamethasone (DEX) and methylprednisolone treatments resulted in dose-dependent inhibition of tumor growth and cell proliferation. Changes in the sensitivity of the clonogenic cell population to 3 mM hydroxyurea were used to assess changes in the clonogenic cell proliferation during and after treatments with DEX or methylprednisolone. Neither methylprednisolone nor DEX given every 12 hr for three doses resulted in significant cell kill in the clonogenic fraction. However, changes in the hydroxyurea sensitivity of the clonogenic population after cessation of DEX treatments indicated G1 cell cycle progression delay with transient enrichment of S-phase clonogenic cells 24 to 48 hr after cessation of DEX treatments. The duration of the DEX-induced progression delay and the timing of maximal S-phase cellularity after DEX was directly correlated with the level of glucocorticoid receptors in the treated tumors. Using regrowth delay to assess the efficacy of kinetically directed sequential chemotherapy, the effectiveness of vincristine, given after DEX, was highly sequence dependent, with the most effective treatment interval being coincident with maximal S-phase clonogenic fraction. Other studies indicated that the effectiveness of cyclophosphamide could also be increased by time sequencing after DEX.

Correlation between glucocorticoid receptor content and the antiproliferative effect of dexamethasone in experimental solid tumors.

The present studies were conducted to assess the antiproliferative effects of dexamethasone (DEX) in murine, rat, and xenograft tumor models and to determine if this kinetic response could be correlated with the level of cytosolic glucocorticoid receptors. Saturable DEX receptors were determined by the dextran-coated charcoal competitive-binding assay, and the antiproliferative effects of DEX were determined by serial measurements of [3H]thymidine labeling indices before and after DEX treatments. The results from such studies in 3 colon tumor lines, one mouse (CO-38) and 2 human xenograft lines (LoVo, H81-4), were qualitatively similar. [3H]Thymidine labeling indices, initially reduced 50 to 60% by the DEX treatments, subsequently increased to as much as 2 times control values, 24 to 36 hr later. With the R3230AC rat mammary tumor model, the DEX dose-dependent antiproliferative effect was similar regardless of whether the tumor was grown in its syngeneic host, Fischer 344 rats, or as a xenograft in athymic nude mice. Studies using the B16 melanoma and SaD2 sarcoma tumor models indicated that the in vivo efficacy of vincristine, given after DEX, was highly sequence dependent. The results from these and other studies in glucocorticoid receptor-positive solid tumor models indicated that the DEX dose-dependent antiproliferative effect and the timing for maximal post-DEX [3H]thymidine labeling indices can be directly correlated with the level of saturable glucocorticoid receptors.

Role of corticosteroid hormones in the control of cell proliferation in residual tumor after surgical cytoreduction.

Changes in tumor cell proliferation in local and distant residual tumor were studied after subtotal surgical cytoreduction in three experimental tumor models varying in corticosteroid receptor content, cell proliferation, and animal host. In residual s.c. RIF-1 anf R3230AC tumors, proliferation was inhibited within 24 hr after 75% resection. Subsequently, intervals of increased proliferation, characterized by increases in tritiated thymidine [( 3H]-dThd) labeling index, primer-dependent DNA polymerase labeling indices, and S-phase clonogenic fractions, were observed. In RIF-1 "artificial" lung metastases. [3H]dThd uptake in tumor-bearing lungs increased by about 70% at 3 days after amputation of "primary" tumor-bearing legs. When dexamethasone was given every 12 hr during the postsurgical recovery interval, changes in [3H]dThd labeling indices and [3H]dThd uptake per lung indicated that the proliferative recovery was delayed until after cessation of dexamethasone treatments. Other studies with RIF-1 tumors indicated that postsurgical tumors indicated that postsurgical proliferation inhibition was dependent on intact adrenal function and that the initiation of postsurgical proliferative recovery was preceded by reestablishment of normal serum corticosterone levels and presurgical levels of saturable glucocorticosteroid receptor. The effectiveness of cyclophosphamide 5-fluorouracil after surgery was time dependent in residual local and distant tumor, with the most efficacious intervals being coincident with postsurgical proliferative recovery. Our results indicate that, in these experimental tumor models, changes in endogenous corticosteroid hormones resulting from the surgical trauma, cellular corticosteroid hormone receptor levels and cytoreduction may influence the time course of the proliferative response in residual tumor after surgical cytoreduction.

In vitro labeling and gold activation autoradiography for determination of labeling index and DNA synthesis times of solid tumors.

In vitro labeling and gold activation autoradiography were used to determine the [3H]thymidine ([3H]TdR)-labeling indices and DNA synthesis times for C3H/He spontaneous mammary tumors. Three variations of the [3H]TdR, [14C]thymidine ([14C]TdR) double-labeling method, together with double-emulsion autoradiography, were used to determine the DNA synthesis times (TS). Tumors labeled totally in vivo (in vivo-in vivo method) and tumors labeled with [3H]TdR in vivo and subsequently labeled with [14C]TdR in vitro showed similar TS values. DNA synthesis times for tumors determined totally in vitro by double labeling (in vitro-in vitro method) were significantly longer than those observed in vivo; however, identical samples subjected to Hypaque-Ficoll gradient separation after double labeling showed TS's similar to those found in vivo. Furthermore, the interval between [3H]TdR and [14C]TdR administration had no effect on TS estimates in vitro. Gold activation autoradiography was used in the present experiments to reduce autoradiographic exposure times. This method, together with in vitro labeling, permits [3H]TdR labeling index and TS determinations after 6-hr and 7-day exposures, respectively.