Red cell volume measurement - using indium as a replacement for chromium.

The withdrawal of Cr-chromate has meant that the technique commonly used for direct measurement of red cell volume has had to be replaced. Most centres moved to a Tc erythrocyte label, however, Tc is known to dissociate over time. We have investigated an alternative technique using an In-chloride and tropolone solution and tested this both in vitro and in vivo. Initial in-vitro and in-vivo work, which included a check of the stability of the radio-labelled product at one hour, demonstrated this label to be stable over this time period. To date, 20 patients have undergone this technique and results show that this technique is a viable alternative to Cr-chromate particularly for patients with splenomegaly who require late sampling. This procedure is now in routine use in our institution.

Rapid decline in 51Cr-ethylenediaminetetraacetic acid-measured renal function during the first weeks following liver transplantation.

BACKGROUND: Renal dysfunction is a serious late complication after liver transplantation (LTX), but there are no studies addressing the early changes associated with this complication. METHODS: We prospectively studied glomerular filtration rate (GFR) before and at 1, 3 and 12 weeks after LTX using 51Cr-labelled ethylenediaminetetraacetic acid clearance in 37 adult consecutive patients who underwent non-acute first LTX. RESULTS: The mean (±SD) age was 49.5 ± 9.5 years, and the male:female sex ratio was 21:16. Diagnoses were autoimmune liver diseases (17), alcoholic cirrhosis (10) and other diseases (10). Immunosuppressive treatment consisted predominantly of triple-drug therapy. A total of 27 of the 37 patients were eligible for GFR analysis at all times. The mean (±SD) GFR was 86 ± 26 mL/min/1.73 m2 before LTX, and 77 ± 30 mL/min/1.73 m2 at 1 week, 64 ± 27 mL/min/1.73 m2 at 3 weeks and 64 ± 23 mL/min/1.73 m2 at 12 weeks after LTX, comparable to a reduction in mean GFR compared with baseline values of 10% (P = 0.1907), 25% (P = 0.0010) and 26% (P = 0.0007). Age and number of blood transfusions during surgery were identified as risk factors for this decline as well as gender, but not pre-transplant diagnosis, model of end-stage liver disease score, cold ischaemia time or post-transplant area under the curve tacrolimus during Days 0-14. CONCLUSIONS: Using measured rather than estimated GFR, our results show that severe renal impairment occurs during the first week after LTX. These results emphasize the need for more studies addressing renoprotective treatment strategies.

Improvement of in vitro potency assays by a resting step for clinical-grade chimeric antigen receptor engineered T cells.

BACKGROUND: Chimeric antigen receptor engineered T (CAR-T) cell therapy is a promising approach currently revolutionizing the field of cancer immunotherapy. However, data concerning clinical-grade CAR-T cell stability and functionality after months of cryopreservation have not been released by companies so far. To investigate the effect of cryopreservation on CAR-T cells and to further optimize the potency assays, we performed this study. METHODS: A third generation of CD19 CAR-T cells was manufactured according to Good Manufacturing Practice (GMP) requirements, which is applied to patients in an ongoing clinical phase 1 study. Quality control tests for sterility, endotoxin and mycoplasma were performed for each batch. Stability in terms of viability, recovery, transduction efficiency and functional capacity was determined using microscopy, multiparametric flow cytometry as well as chromium-51 release tests. RESULTS: Up to 90days of cryopreservation had no influence on viability, recovery and transduction efficiency of CAR-T cells. However, higher cell concentration for cryopreservation could alter the cell viability and recovery but not the transduction efficiency. Moreover, directly after thawing, both the quantity and quality of the functionality of CAR-T cells were transiently hampered by the negative effects of cryopreservation. Notably, the impaired functionality could be fully restored and even strengthened after an overnight resting process. DISCUSSION: Cryopreservation is a challenge for the functional activity of CAR-T cells. However, CAR-T cells regain their potency by overnight incubation at 37°C, which mimics the clinical application setting. Therefore, an overnight resting step should be included in in vitro potency assays.

Evaluating Antibody-Dependent Cell-Mediated Cytotoxicity by Chromium Release Assay.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism in which immune cell activation is induced by the cross-linking of CD16 with the Fc region of antibodies that at the same time bind specifically to cell surface antigens. ADCC stimulates the secretion of perforin, granzymes, and cytokines leading to lysis of the malignant cells. Natural killer (NK) cells express the CD16 receptor and can therefore be activated by ADCC to kill tumor cells. To study the cytotoxicity of NK cells against cancer cells, an ADCC-based assay is described: the chromium release assay. In this method, the antibody trastuzumab, which binds specifically to HER2-positive malignant cells, is used to trigger ADCC.

Further studies to evaluate methods of leucoreduction to prevent alloimmune platelet refractoriness and induce tolerance in a dog platelet transfusion model.

OBJECTIVES: Three leucoreduction filters were evaluated - when used alone or combined with centrifuge leucoreduction (C-LR) - to prevent alloimmune platelet refractoriness in a dog platelet transfusion model. MATERIALS AND METHODS: Donor platelet-rich plasma (PRP) or buffy coat (BC) platelets were either filter leucoreduced (F-LR) or F-LR/C-LR, (51) Cr radiolabelled and transfused. Weekly transfusions were given for up to 8 weeks or until platelet refractoriness. Recipients who accepted treated transfusions were then given non-leucoreduced (non-LR) platelets to determine whether donor-specific tolerance had been induced. RESULTS: Acceptance of F-LR PRP transfusions ranged from 29% to 66%. F-LR/C-LR transfusions prepared from PRP were accepted by 92%, from BC by 63% and from pooled PRP by 75% of recipients (p=NS); overall acceptance rate of F-LR/C-LR transfusions was 83%. Tolerance to subsequent non-LR transfusions occurred in 45% of the F-LR-/C-LR-accepting recipients unrelated to DR-B compatibility between donors and recipients (P = 0·18). CONCLUSION: In a dog platelet transfusion model, acceptance of donor platelets required combining F-LR with C-LR as apparently each process removes different immunizing WBCs.

Measuring cytotoxicity by bioluminescence imaging outperforms the standard chromium-51 release assay.

The chromium-release assay developed in 1968 is still the most commonly used method to measure cytotoxicity by T cells and by natural killer cells. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Since then, alternative methods have been developed using different markers of target cell viability that do not involve radioactivity. Here, we compared and contrasted a bioluminescence (BLI)-based cytotoxicity assay to the standard radioactive chromium-release assay using an identical set of effector cells and tumor target cells. For this, we stably transduced several human and murine tumor cell lines to express luciferase. When co-cultured with cytotoxic effector cells, highly reproducible decreases in BLI were seen in an effector to target cell dose-dependent manner. When compared to results obtained from the chromium release assay, the performance of the BLI-based assay was superior, because of its robustness, increased signal-to-noise ratio, and faster kinetics. The reduced/delayed detection of cytotoxicity by the chromium release method was attributable to the association of chromium with structural components of the cell, which are released quickly by detergent solubilization but not by hypotonic lysis. We conclude that the (BLI)-based measurement of cytotoxicity offers a superior non-radioactive alternative to the chromium-release assay that is more robust and quicker to perform.

An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.

The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.

Novel interaction between proliferating cell nuclear antigen and HLA I on the surface of tumor cells inhibits NK cell function through NKp44.

NK cell function is closely regulated by numerous inhibitory and activating receptors binding corresponding ligands on the surface of target cells, providing vital first line defenses against infections and cancer. NKp44, originally discovered as an activating NK cell receptor, was recently found to elicit inhibitory effects on NK cell effector function through recognition of cell surface PCNA. Other reports have pointed to potential associations between NKp44 and HLA I molecules, as well as HLA I and Damage Associated Molecular Pattern molecules (DAMPs) on the surface of tumor cells. In this report, we have identified novel interaction between HLA I and PCNA on the surface of human tumor cells by confocal microscopy and immunoprecipitation. In addition to previous reports, we show PCNA on the cell surface where novel association with HLA I does not require the presence of NKp44 expressing NK cells and occurs with endogenous PCNA. The association of HLA I and PCNA forms the inhibitory ligand for NKp44, resulting in inhibition of NK cell cytotoxicity. We further postulate NCR ligands are composed of DAMP molecules localized to the cell surface, colocalizing with HLA I, and potentially heparin sulfate proteoglycans.

The MTT assay is a rapid and reliable quantitative method to assess Staphylococcus aureus induced endothelial cell damage.

We established the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to assess damage induced by Staphylococcus aureus in human umbilical vein endothelial cells (HUVECs). The MTT assay was comparable to the previously published (51)chromium release assay. MTT reduction by intracellular S. aureus was negligible and therefore permits assessment of S. aureus induced EC damage.

Evaluating CD8⁺ T cell responses in vitro.

The (51)Cr-release assay described in the 1960s has been for decades the gold standard cytolytic assay and remains in use in many laboratories. Whereas other radioactive tests were later on described, they never fully replaced the (51)Cr-release assay. More thorough understanding of CTL biology and killing pathways has more recently resulted in the design of reliable nonradioactive tests to analyze CD8(+) T cell responses which are likely to supplant in a close future the (51)Cr-release assay.

Roles of crustacean hyperglycaemic hormone in ionic and metabolic homeostasis in the Christmas Island blue crab, Discoplax celeste.

There is a growing body of evidence implicating the involvement of crustacean hyperglycaemic hormone (CHH) in ionic homeostasis in decapod crustaceans. However, little is known regarding hormonally influenced osmoregulatory processes in terrestrial decapods. As many terrestrial decapods experience opposing seasonal demands upon ionoregulatory physiologies, we reasoned that these would make interesting models in which to study the effect of CHH upon these phenomena. In particular, those (tropical) species that also undergo seasonal migrations might be especially informative, as we know relatively little regarding the nature of CHHs in terrestrial decapods, and hormonally mediated responses to seasonal changes in metabolic demands might also be superimposed or otherwise integrated with those associated with ionic homeostasis. Using Discoplax celeste as a model crab that experiences seasonal extremes in water availability, and exhibits diurnal and migratory activity patterns, we identified two CHHs in the sinus gland. We biochemically characterised (cDNA cloning) one CHH and functionally characterised (in terms of dose-dependent hyperglycaemic responses and glucose-dependent negative feedback loops) both CHHs. Whole-animal in situ branchial chamber (22)NaCl perfusion experiments showed that injection of both CHHs increased gill Na(+) uptake in a seasonally dependent manner, and (51)Cr-EDTA clearance experiments demonstrated that CHH increased urine production by the antennal gland. Seasonal and salinity-dependent differences in haemolymph CHH titre further implicated CHH in osmoregulatory processes. Intriguingly, CHH appeared to have no effect on gill Na(+)/K(+)-ATPase or V-ATPase activity, suggesting unknown mechanisms of this hormone's action on Na(+) transport across gill epithelia.

In vivo muscle electroporation threshold determination: realistic numerical models and in vivo experiments.

In vivo electroporation is used as an effective technique for delivery of therapeutic agents such as chemotherapeutic drugs or DNA into target tissue cells for different biomedical purposes. In order to successfully electroporate a target tissue, it is essential to know the local electric field distribution produced by an application of electroporation voltage pulses. In this study three-dimensional finite element models were built in order to analyze local electric field distribution and corresponding tissue conductivity changes in rat muscle electroporated either transcutaneously or directly (i.e., two-plate electrodes were placed either on the skin or directly on the skeletal muscle after removing the skin). Numerical calculations of electroporation thresholds and conductivity changes in skin and muscle were validated with in vivo measurements. Our model of muscle with skin also confirms the in vivo findings of previous studies that electroporation "breaks" the skin barrier when the applied voltage is above 50 V.

Endogenous cannabinoid system regulates intestinal barrier function in vivo through cannabinoid type 1 receptor activation.

The deleterious effects of stress on the gastrointestinal tract seem to be mainly mediated by the induction of intestinal barrier dysfunction and subsequent subtle mucosal inflammation. Cannabinoid 1 receptor (CB1R) is expressed in the mammalian gut under physiological circumstances. The aim of this investigation is to study the possible role of CB1R in the maintenance of mucosal homeostasis after stress exposure. CB1R knockout mice (CB1R(-/-)) and their wild-type (WT) counterparts were exposed to immobilization and acoustic (IA) stress for 2 h per day during 4 consecutive days. Colonic protein expression of the inducible forms of the nitric oxide synthase and cyclooxygenase (NOS2 and COX2), IgA production, permeability to (51)Cr-EDTA, and bacterial translocation to mesenteric lymph nodes were evaluated. Stress exposure induced greater expression of proinflammatory enzymes NOS2 and COX2 in colonic mucosa of CB1R(-/-) mice when compared with WT animals. These changes were related with a greater degree of colonic barrier dysfunction in CB1R(-/-) animals determined by 1) a significantly lower IgA secretion, 2) higher paracellular permeability to (51)Cr-EDTA, and 3) higher bacterial translocation, both under basal conditions and after IA stress exposure. Pharmacological antagonism with rimonabant reproduced stress-induced increase of proinflammatory enzymes in the colon described in CB1R(-/-) mice. In conclusion, CB1R exerts a protective role in the colon in vivo through the regulation of intestinal secretion of IgA and paracellular permeability. Pharmacological modulation of cannabinoid system within the gastrointestinal tract might be therapeutically useful in conditions on which intestinal inflammation and barrier dysfunction takes place after exposure to stress.

Naegleria fowleri lysate induces strong cytopathic effects and pro-inflammatory cytokine release in rat microglial cells.

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A (51)Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-α, IL-6, and IL-1ß, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

Correlation of various published radionuclide glomerular filtration rate estimation techniques and proposed paediatric normative data.

OBJECTIVE: The aim of this study is to assess the comparability and interchangeability of the radionuclide glomerular filtration rate (GFR) using different published techniques, and propose normative data for paediatrics. METHODS: A total of 476 paediatric oncology patients aged 2-17 years, referred between January 2001 and December 2008 for GFR estimation, were reviewed for any potential cause of renal impairment. Sixty-nine patients met the stringent inclusion criteria, and were included in the study. GFR estimation was carried out using either technetium-99m diethylene triamine penta-acetic acid (99mTc-DTPA) or chromium-51 EDTA (5¹Cr-EDTA). Multiple GFR results were calculated from the same blood sample data (counts/min/ml), according to previously published GFR estimation techniques using one to three blood samples. These techniques were slope-intercept, slope-only and half life. For slope-intercept techniques, GFR was normalized to body surface area or extracellular fluid volume. RESULTS: The GFR values obtained using different techniques were highly variant. The intraclass correlation (ICC) for different methods was moderate (ICC=0.56-0.66). A reliable empiric formula to allow conversion of GFR values from one technique to another could not be derived because of this variability, with some exceptions. 5¹Cr-EDTA yielded the same or lower variability than 99mTc-DTPA. The British Nuclear Medicine Society-recommended method had the lowest coefficient of variation, with a mean value of 116 (SD 22) normalized to 1.73 m² for 5¹Cr-EDTA using two samples. CONCLUSION: The GFR values obtained from different calculation techniques are not readily interchangeable or comparable, with some exceptions. For both 99mTc-DTPA and 5¹Cr-EDTA, the British Nuclear Medicine Society-recommended technique appears to be the most robust, with the least coefficient of variation.

Assessment of permeability barriers to macromolecules in the rodent endometrium at the onset of implantation.

In rodents, embryo implantation is an invasive process, which begins with its attachment to the uterine wall and culminates in the formation of the definitive placenta several days later. It is critical that the endometrium provide a supportive environment for the implanting embryo during this process, as the placenta is not yet established. The concept of changing permeability barriers to macromolecules between different extracellular compartments in the rodent uterus at the onset of implantation has been established. This chapter provides protocols that can be used to assess this changing permeability barrier and the associated redistribution of macromolecules during the early phases of implantation in rodents. An increased permeability of the endometrial vasculature to plasma proteins occurs in areas adjacent to the implanting blastocyst. In addition, alterations in the extracellular matrix enhance the accumulation of fluid and extravasated macromolecules. We describe several protocols proven to be effective in studying and quantifying early vascular and extravascular responses to natural and artificial "implantation stimuli." The first three protocols represent qualitative and quantitative methods to assess the early endometrial "vascular permeability" response. On the contrary, the fourth protocol addresses the onset of decidualization and the arising permeability barrier, which restricts the movement of macromolecules through the extracellular space. This barrier is believed to provide transient protection for the implanting embryo against potentially harmful maternal serum proteins. This protocol describes assessment of resistance of the primary decidual zone to the movement of macromolecules across the compartments of the extracellular space.

Naegleria fowleri Lysate Induces Strong Cytopathic Effects and Pro-inflammatory Cytokine Release in Rat Microglial Cells

Naegleria fowleri, a ubiquitous free-living ameba, causes fatal primary amebic meningoencephalitis in humans. N. fowleri trophozoites are known to induce cytopathic changes upon contact with microglial cells, including necrotic and apoptotic cell death and pro-inflammatory cytokine release. In this study, we treated rat microglial cells with amebic lysate to probe contact-independent mechanisms for cytotoxicity, determining through a combination of light microscopy and scanning and transmission electron microscopy whether N. fowleri lysate could effect on both necrosis and apoptosis on microglia in a time- as well as dose-dependent fashion. A 51Cr release assay demonstrated pronounced lysate induction of cytotoxicity (71.5%) toward microglial cells by 24 hr after its addition to cultures. In an assay of pro-inflammatory cytokine release, microglial cells treated with N. fowleri lysate produced TNF-alpha, IL-6, and IL-1beta, though generation of the former 2 cytokines was reduced with time, and that of the last increased throughout the experimental period. In summary, N. fowleri lysate exerted strong cytopathic effects on microglial cells, and elicited pro-inflammatory cytokine release as a primary immune response.

No difference in Gag and Env immune-response profiles between vaccinated and non-vaccinated rhesus macaques that control immunodeficiency virus replication.

Recent advances in human immunodeficiency virus (HIV) vaccine design have resulted in induction of strong CD4 T-cell proliferative and polyfunctional cytokine responses, which are also characteristic for long-term non-progressing (LTNP) HIV-infected individuals. However, limited information is available on the persistence of these responses after infection. Results from studies in non-human primates indicate that vaccine-induced immune responses are partially maintained upon viral infection and differ from the responses seen in non-vaccinated animals that typically progress to disease. However, it is unclear how these partially preserved responses compare to immune responses that are acquired naturally by LTNP animals. In this study, immune-response profiles were compared between vaccinated animals that, upon SHIV89.6 challenge, became infected but were able to control virus replication, and a group of animals having spontaneous control of this viral infection. Both groups were found to develop very similar immune responses with regard to induction of CD4 and CD8 T-cell polyfunctional cytokine responses, proliferative capacity and cytotoxic capacity, as measured by a standard 51Cr release assay and more direct ex vivo and in vivo CTL assays. Hence, vaccinated animals that become infected, but control infection, appear to establish immune responses that are similar to those elicited by long-term non-progressors.

Studying NK cell responses to ectromelia virus infections in mice.

Here we describe methods for the in vivo study of antiviral NK cell responses using the mouse Orthopoxvirus ectromelia virus as a model, the agent of mousepox. The methods include those specific for the preparation and use of ectromelia virus such as the production of virus stocks in tissue culture and in live mice, the purification of virus stocks, the titration of virus stocks and virus loads in organs, and the infection of mice. The chapter also includes methods for the specific study of NK cell responses in infected mice such as the preparation of organs (lymph nodes, spleen, and liver) for analysis, the study of NK cell responses by flow cytometry, the adoptive transfer of NK cells, the measurement of NK cell cytolytic activity ex vivo and in vivo, and the determination of NK cell proliferation by bromodeoxyuridine loading or by dilution of carboxyfluorescein diacetate succinimidyl ester (CFSE).

Stable expression of EBV-gp350 on the surface of NC37 cells confers natural killer (NK)-cell susceptibility or resistance, depending on the assay used to assess NK-mediated function.

NC37 cells containing the Epstein-Barr virus (EBV) genome do not express the viral glycoprotein-350 (gp350) on the cell surface. Despite being a cancer cell line, NC37 cells show resistance to natural killer (NK) cell cytotoxicity by the standard chromium ((51)Cr) release assay (CRA). EBV-gp350 has been identified as a ligand for antibody dependent cell-mediated cytotoxicity (ADCC). The stable expression of gp350 on the NC37 cell surface membrane could make this cell line a suitable target for measuring ADCC antibody. The pcDNA3.1-gp350 was transfected into the stably expressing enhanced green fluorescent protein (EGFP)-NC37 cell line. The transfected cells were then selected for expression of gp350 on the cell surface using immunomagnetic bead-based sorting. The gp350-EGFP-NC37 cell line was then re-examined for resistance to NK cytotoxicity, and compared with the standard K562 and EGFP-K562 cell lines using the CRA and a flow cytometric method, respectively. Surprisingly, the gp350-EGFP-NC37 cells, like the parental NC37 cell line, showed comparable resistance to NK cell-mediated cytotoxic activity by the CRA, while demonstrating susceptibility to NK cell cytotoxicity comparable to EGFP expressing K562 cells by the flow cytometric method. The susceptibility of gp350-EGFP-NC37 cells to NK cell cytotoxic activity is dependent on the type of assay.