Increase In Positive Cells Research Articles

Abstract 2810: Increasing the efficacy of KRASG12Cinhibitor therapy through the adjuvant use of FAP targeted radiotherapy in a murine model of lung cancer

Abstract The purpose of this study was to evaluate the combined effects of KRAS targeted therapy and FAP targeted radiotherapy compared to either therapy alone in an animal model of lung cancer. KRAS is a protein that acts as a molecular switch regulating cell proliferation mediated through binding of GTP versus GDP. When mutated, the switch remains “on” and cells undergo unrestrained proliferation. KRAS mutations are frequently found in lung cancer and it appeared for years as if they could not be inhibited. Recently KRASG12C inhibitors have been developed by targeting a site adjacent to the binding pocket that induces a conformational change in the protein preventing GTP binding thus inhibiting unrestrained cell cycling. While a promising advance, KRAS targeted therapy does not confer a durable response and therefore it is essential to develop adjuvant therapies that enhance efficacy of KRAS targeting agents. FAP targeted radiotherapy is a new anticancer therapeutic that delivers a radioactive payload directly to tumors and has been shown to increase efficacy when used with immunomodulatory therapy. We studied the effect of KRAS targeted therapy alone and in combination with FAP targeted radiotherapy. Animals were implanted with KRASG12C heterozygote LLC1 lung cancer cells and treated according to the following groups: untreated control, sotorasib treatment, 177Lu-FAPI-04 treatment, and combined treatment. Tumor growth was evaluated every 2-3 days and animals were euthanized at 21 days and tumors extracted for analysis. anti-Ki67 and anti-caspase-3 immunofluorescence assessed cell cycling and apoptotic cell death, respectively. Immunoassays were performed to quantify soluble analytes, including IL-12p70, TNF-α, TGF-β1, and IFN-γ. Tumor growth in dual treated animals was significantly reduced compared to either therapy alone. Ki67 immunofluorescence demonstrated a significantly decreased number of cells in the combined therapy group than either therapy alone (p=0.01 and 0.02 when compared to sotorasib alone and radiotherapy alone, respectively). Anti-caspase-3 immunofluorescence demonstrated a significant increase in positive cells in the combined group compared to animals treated with sotorasib alone (p=0.003). Immunoassays assessing expression of TNF-α, TGF-β1, and IFN-γ from tumors from animals in each treatment group demonstrated statistically significant differences (p=0.006, p=0.03, p=0.02, respectively). Combined KRAS targeted therapy and FAP targeted radiotherapy results in decreased tumor growth compared to either therapy alone. Effects are mediated through decreased cell cycling, increased apoptotic cell death, and changes in key cell signaling pathways. Future translational studies will assess this therapy combination in patients to evaluate its ability to increase response to therapy. Citation Format: Kathleen M. Capaccione, Mikhail Doubrovin, Brian Braumuller, Sophia Huang, Nikunj Bhatt, Fatemeh Momen-Heravi, Andrei Molotkov, Michael Kissner, Alessandra Ali, Akiva Mintz. Increasing the efficacy of KRASG12Cinhibitor therapy through the adjuvant use of FAP targeted radiotherapy in a murine model of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2810.

IFIT5 Participates in the Antiviral Mechanisms of Rainbow Trout Red Blood Cells

Viral hemorrhagic septicemia virus (VHSV) infection appears to be halted in rainbow trout nucleated red blood cells (RBCs). Diverse mechanisms are thought to be related to the antiviral immune response of rainbow trout RBCs to VHSV. However, the specific rainbow trout RBC proteins that interact directly with VHSV are still unknown. In an attempt to identify VHSV-RBC protein interactions, we characterized the immunoprecipitated (IP) proteome of RBCs exposed to VHSV using an antibody against the N protein of VHSV. The IP proteomic characterization identified 31 proteins by mass spectrometry analysis. Among them, we identified interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), a protein belonging to a family of proteins that are induced after the production of type I interferon. Importantly, IFIT5 has been implicated in the antiviral immune response. We confirmed the participation of IFIT5 in the rainbow trout RBC antiviral response by examining the expression profile of IFIT5 in RBCs after VHSV exposure at transcriptional and protein levels. We detected a correlation between the highest IFIT5 expression levels and the decline in VHSV replication at 6 h post-exposure. In addition, silencing ifit5 resulted in a significant increase in VHSV replication in RBCs. Moreover, an increase in VHSV replication was observed in RBCs when the IFIT5 RNA-binding pocket cavity was modulated by using a natural compound from the SuperNatural II database. We performed a proximity ligation assay and detected a significant increase in positive cells among VHSV-exposed RBCs compared to unexposed RBCs, indicating protein-protein colocalization between IFIT5 and the glycoprotein G of VHSV. In summary, these results suggest a possible role of IFIT5 in the antiviral response of RBCs against VHSV.

Persistent Primary Cytomegalovirus Infection After Deceased Donor Kidney Transplant: Ganciclovir Susceptibility of Human Cytomegalovirus With UL97 D605E Mutation: A Case Report

BackgroundCytomegalovirus (CMV) could cause rejection in immunocompromised patients during early post-renal transplant stage. The American Transplant Society guidelines recommend prophylactic therapy with ganciclovir (GCV) for 3 to 6 months to prevent CMV infections in adult renal transplant patients. However, there is no recommended CMV treatment regimen for pediatric patients. Main FindingsWe performed deceased donor kidney transplant from an anti-CMV antibody-positive donor to an anti-CMV antibody-negative 15-year-old female recipient with end-stage renal disease caused by bilateral renal hypoplasia. One month after transplant, increase in positive cells in the CMV antigenemia assay indicated a primary CMV infection in the patient, who immediately received GCV. She was switched to foscarnet after 4 months of anti-CMV therapy because of clinical GCV resistance. CMV was isolated from the peripheral blood mononuclear cells but neutralizing antibody was not detected. Isolated CMV was susceptible to GCV and foscarnet, although it carried the UL97 D605E mutation, assumed to be associated with GCV resistance. ConclusionsThe primary CMV infection presented a phenotypic clinical drug resistance, but all recovered CMV isolates were drug-susceptible even if isolated after prolonged anti-CMV therapy, indicating that immune status was more important for recovery from primary CMV infection than anti-CMV therapy.

Disruption of redox homeostasis in liver function and activation of apoptosis on consumption of aspartame in folate deficient rat model

This study assesses the effect of long-term intake of aspartame on liver function and apoptosis signaling pathway in the Wistar albino rats. Several reports have suggested that methanol is one of the major metabolites of Aspartame. Non-primate animals are usually resistant to methanol-induced metabolic acidosis due to high levels of hepatic folate content; hence a folate deficiency model was induced by treating animals with methotrexate (MTX) prior to aspartame exposure. The aspartame treated MTX animals exhibited a marked significant increase in hepatic alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and lactic acid dehydrogenase (LDH) activity compared to controls. Aspartame treated MTX animals additionally exhibited down-regulation of genes namely B-cell lymphoma 2 (Bcl2) expression and up-regulation of Bcl-2-associated X protein (Bax), Fas-associated protein with death domain (FADD) and Caspase 3, 9 genes and apoptotic protein expression, indicating the augmentation of hepatic apoptosis. Nuclear condensation, micro vacuole formation in the cytoplasm and necrosis were observed in the liver of the aspartame treated animals on histopathology evaluation. Additionally, Immunohistochemical analysis revealed a significant increase in positive cells expressing Fas, FADD, Bax and Caspase 9 protein, indicating an increase in apoptotic protein expression in the liver. Thus, Aspartame may act as a chemical stressor which alters the functional status of liver, leading to hepatotoxicity.

Pubertal exposure to ethinylestradiol promotes different effects on the morphology of the prostate of the male and female gerbil during aging.

In rodents, the final growth and maturation of the prostate occur at puberty, a crucial period for prostate development. The present study is a serological, morphological, morphometric, and immunohistochemical analysis of the effects of exposure to ethinylestradiol (EE) (15 µg/kg/day) during puberty (EE/PUB group) on the male ventral and female prostate in senile gerbils. In the study, male and female gerbils (Meriones unguiculatus) (42 days) received by gavage 15 μg/kg/day of EE (a component of the contraceptive pill), diluted in 100 µL of Nujol® for 1 week (EE/PUB group). In the control group, males and females were not treated. Animals were killed (n = 5) after 12 months in the experimental groups. In the senile male in the EE/PUB group, we observed a reduction in testosterone levels and a decrease in the prostatic epithelial thickness, as well as in the thickness of the muscle layer. In addition, an increase in PIN multiplicity and prostatic inflammation was observed. In the senile female in the EE/PUB group, we observed increased testosterone and estradiol levels, an enhanced prostatic epithelial thickness and an increase in the thickness of the muscle layer. Immunohistochemical analysis revealed an increase in positive cells (%) for AR and PCNA in the male prostate and an increase in positive basal cells for p63 in the female prostate of the EE/PUB group. Exposure to EE during puberty resulted in an inhibitory action on the male ventral prostate and an anabolic effect on the female prostate in senile gerbils. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 477-489, 2017.

In vivo genotoxicity of Ginkgo biloba extract in gpt delta mice and constitutive androstane receptor knockout mice.

The National Toxicology Program study of Ginkgo biloba extract (GBE), a herbal supplement, reported concerns regarding genotoxicity and clear evidence of hepatocarcinogenicity and liver hypertrophy in mice. To clarify the genotoxicity of GBE in vivo, we performed reporter gene mutation assay using gpt delta mice. We also used a combined liver comet assay and bone marrow micronucleus assay using C3H-derived constitutive androstane receptor knockout (CARKO) and wild-type mice. No remarkable increases in gpt or Spi(-) mutation frequencies were observed in DNA extracted from the livers of gpt delta mice that had been exposed to GBE up to 2000 mg/kg bw/day. In the comet and micronucleus assays, no statistically significant increases in positive cells were observed at doses up to 2000 mg/kg bw/day of GBE in either mouse genotype. The present study provides clear evidence that GBE is not genotoxic in vivo. Our results indicate that GBE-induced hepatocarcinogenesis in mice occurs through a non-genotoxic mode of action.

Overexpression of regenerating gene Iα appears to reflect aberration of crypt cell compartmentalization in sessile serrated adenoma/polyps of the colon

BackgroundColorectal sessile serrated adenoma/polyps (SSA/Ps) are characterized by asymmetrical distribution of Ki67-positive cells, which varies among crypts and involves the crypt length to a variable extent; the pattern has been designated as aberration of crypt cell compartmentalization. The regenerating gene (REG) Iα is a cell growth and/or anti-apoptotic factor and its overexpression might be associated with aberration of crypt cell compartmentalization in SSA/Ps. We investigated REG Iα expression in SSA/Ps in comparison to hyperplastic polyps (HPs).MethodsA total of 64 cases of serrated polyps (≥10 mm in size), including 53 SSA/Ps and 11 HPs, were included in the present study. Immunostaining was performed using a labeled streptavidin-biotin method. REG Iα expression was classified as follows: (i) expression of endocrine cells: grade 0 (a few positive cells) to 3 (marked increase in positive cells); (ii) expression of goblet cells: grade 0 (negative) to 2 (positive for crypts and surface epithelial cells); (iii) staining intensity of goblet cells: grade 0 (negative) to 2 (strong); (iv) staining intensity of crypt (absorptive) cell membranes: grade 0 (negative) to 2 (strong). The presence of aberration of crypt cell compartmentalization was assessed using Ki67 immunostaining.ResultsWith regard to the REG Iα expression of endocrine cells, 8 out of 11 HPs (73%) were grade 0, whereas 51 of 53 SSA/Ps (96%) were grade 1 or higher (p < 0.001). With regard to the distribution of REG Iα-immunoreactive goblet cells, 10 of 11 HPs (91%) were grade 1, whereas 50 of 53 SSA/Ps (94%) were grade 2 (p < 0.001). A similar trend was found in the staining intensity of goblet cells or crypt cell membranes (p = 0.011). Aberration of crypt cell compartmentalization was more frequently identified in SSA/Ps (72%) than in HPs (18%; p = 0.002). A significant association was observed between REG Iα overexpression and the aberration of crypt cell compartmentalization in serrated polyps (p = 0.037).ConclusionsREG Iα overexpression is a characteristic of SSA/Ps, which appears to reflect aberration of crypt cell compartmentalization.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7240956081100040

Response to Alzheimer&amp;rsquo;s research paper

Dear editor Zhang et al have recently published an interesting article in Clinical Interventions in Aging entitled “Atorvastatin attenuates the production of IL-1β, IL-6, and TNF-α in the hippocampus of an amyloid β1-42-induced rat model of Alzheimer’s disease”.1 The conclusion that “IL-1β, IL-6, and TNF-α were significantly decreased in the atorvastatin-treated AD group than that in the AD group” is not justified by the data presented in Figures ​Figures1,1, ​,2,2, and ​and3.3. Given that the AD group and the atorvastatin-treated AD group showed no significant difference in numbers of cells positive for interleukin-1β, interleukin-6, or tumor necrosis factor-α, and in some cases there may be a trend towards an increase in positive cells in the atorvastatin-treated AD group, it is hard to see how they came to this conclusion. Figure 3 Atorvastatin attenuated interleukin-1β expression in the hippocampus of a rat treated with Aβ1-42. The upper panel shows interleukin-1β-positive cells in rat hippocampus, detected by immunohistochemistry on day 7 after Aβ ... Figure 4 Atorvastatin attenuated interleukin-6 expression in the hippocampus of a rat treated with Aβ 1-42. The upper panel shows interleukin-6-positive cells in rat hippocampus, detected by immunohistochemistry on day 7 after Aβ injection (original ... Figure 5 Atorvastatin attenuated TNF-α expression in hippocampus of the Aβ1-42-treated rat. The upper panel shows TNF-α-positive cells in rat hippocampus, detected by immunohistochemistry on day 7 after Aβ injection (original magnification ... In their introduction, Li et al state that “Cholesterol-lowering agents, particularly 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (also known as statins), have been reported to be capable of increasing the ratio of alpha to beta secretase activity and then increasing the concentrations of extracellular Aβ.” The conventional wisdom, supported by the references that they cite (eg, Buxbaum et al2), is that changing the ratio in this way is likely to lead to a decrease, not an increase, in extracellular Aβ. I would very much like to hear the authors’ and your views on these issues.

Enteroendocrine cells and appetite dysregulation in Crohn's disease

IntroductionLoss of appetite and malnutrition in active Crohn's disease (CD) are important problems. The biological mechanisms underpinning appetite loss are unclear. Enteroendocrine cells (EEC) form a pivotal part of the...

Neuroprotective effects of tempol on retinal ganglion cells in a partial optic nerve crush rat model with and without iron load

Iron overload can contribute to oxidative stress in many tissues. We studied the effects of pretreatment with iron dextran on RGC loss in a calibrated partial optic nerve crush (PONC) model in rats, along with the protection offered by tempol (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl, a membrane-permeable superoxide dismutase mimetic and free-radical scavenger), in the same experimental paradigm. A total of 40 rats in 6 groups of 5–8 animals each underwent PONC in one eye and sham crush in the other. Animals were pretreated with a single iron dextran load 24 h prior to PONC, and treated with tempol 6 h before and then once daily after PONC. Control animals were treated with PBS. RGC were retrogradely labeled with a fluorescent marker; all data are expressed in percent of the RGC count in the respective sham-treated eye. Immunohistochemistry was performed to visualize 3-nitrotyrosine, a marker of nitroxidative stress. PONC without iron pretreatment resulted in the survival of only 31.4% of labeled RGC after 7 days. Even fewer RGC (12.7%) survived after PONC with iron pretreatment. However, tempol in doses of 20 mg/kg of body weight (BW) significantly attenuated this effect when given as described above; in the group without iron pretreatment the number of surviving RGC doubled from 31.4% to 62.1%. In the group with iron pretreatment the survival rate of RGC increased even more pronouncedly, from 12.7% without tempol to 46.2% with tempol. Tempol in doses of 1 mg/kg BW and 5 mg/kg BW showed no significant rescue of RGC. Immunostaining showed nitrotyrosine-positive RGCs in PONC but not in sham-treated eyes and an increase in positive cells after iron load. Tempol treatment reduced nitrotyrosine staining in both the iron and non-iron groups. Our results demonstrate that PONC results in significantly greater RGC damage when iron pretreatment is performed, and that the compound tempol may provide additional protection for RGC in cases of neuronal damage both with and without prior iron treatment.

Role of Nurr1 and Ret in inducing rat embryonic neural precursors to dopaminergic neurons

Background: Nurr1, a member of the nuclear receptor superfamily of transcription factors, is highly expressed in midbrain dopaminergic (DA) neurons. Ret is a member of the receptor tyrosine kinase (RTK) superfamily and a critical signal transducing subunit of receptors for glial cell line-derived neurotrophic factor (GDNF). Both Nurr1 and Ret play important roles in the development of DA neurons.Purpose: To investigate possible correlation between Nurr1 and Ret on inducing expression of tyrosine hydroxylase (TH) in neural precursor cells.Methods: Neural precursors isolated from rat embryonic mesencephalon (E13.5d) were transfected with vectors containing Nurr1 or Ret and treated with 9-cis-retinoic acid (RA) and/or GDNF for 3 days. RT-PCR and immunocytochemistry was used to test the expression of Nurr1 and Ret and the TH positive cells.Results: The number of TH positive cells was increased from 1.53 ± 0.12 to 3.83 ± 0.56% after the cells were transfected with Nurr1. Increased endogenous Nurr1 by RA lead to 1.8 times (2.86 ± 0.32% versus 1.53 ± 0.12% in the controls) increase in TH positive cells. A double inducing effect by both endogenous and exogenous Nurr1 on the expression of TH was observed by 3.3 times increase in the positive cells (from 5.03 ± 0.76 to 1.53 ± 0.12% in control). Ret expression was induced by overexpression of Nurr1. Overexpressed Ret had no inducing effect on the expression of Nurr1 and the number of TH positive cells. The gene of dopamine transporter (DAT) was clearly induced in the cells transfected with Ret.Conclusion: Nurr1, required for the expression of Ret, had inducing effect on TH positive cells, and Ret may promote maturation of DA neuron by up-regulating DAT expression through its ligand. As a cooperator, Ret seems to work together with Nurr1 in the development of DA neurons.

Aberrant Expression Patterns of Histone H3 mRNA, p53, Cyclin D1, and Cyclin B1 in Oral Neoplastic Epithelial Lesions

In this paper, tumor growth was studied using oncogenic and kinetic analysis. In verrucous carcinoma (VC, n=22), squamous papilloma (SP, n=18), and hyperkeratotic lesion (HK, n=12), cellular proliferative potentials were evaluated and compared. Co-expression of histone H3 mRNA and p53 protein was not found in HK. In 6 cases (33.3%) of SP, co-expression was detected within the basal and parabasal layers. However, in 17 cases (77.2%) of VC, co-expression of histone H3 mRNA and p53 protein was found extensively from basal to spinous layers. Cyclin D1 was detected in 5 cases (41.7%) of HK and in 14 cases (77.8%) of SP at parabasal layer. While in 20 cases (90.9%) of VC, cyclin D1 was detected from basal to spinous layers, and positive (2+ and 3+) cases (45.4%) increased. Cyclin B1 was detected in 9 cases (75.0%) of HK and in 16 cases (88.9%) of SP at basal or parabasal layer, and in all 22 cases of VC from basal to spinous layers, and positive (2+ and 3+) cases (68.1%) increased. In the intra-cellular distribution of cyclin B1, there were many cases of cytoplasmic types (66.7%) in HK, of cytoplasmic dominant types (50.0%) in SP, and of cytoplasmic dominant types (72.7%) in VC. There were also some cases of nuclear dominant types (27.3%) in VC. The present study suggests that increase of p53 protein, increase of cell proliferation, wide epithelial distribution of cyclins D1 and B1, and increase of positive cells in the nucleus with cyclin B1 were indicators of malignant potential.

Nitric oxide synthase in tissues around failed hip prostheses

Nineteen patients who had undergone hip revision surgery for aseptic loosening of joint prostheses were studied. Tissue samples were harvested at the interface between bone and implant, either at the stem or at the cotyle level. Immunohistochemistry was performed on tissue sections to detect nitric oxide synthase (NOS), the enzyme which enables the synthesis of nitric oxide (NO), a molecule which can activate bone resorption. Quantitative analysis of the positive cells and correlation with the presence of particulate wear debris and radiological data were performed. The authors observed a trend towards a moderate increase in positive cells due to inducible NOS in tissues containing particulate wear debris, especially of a plastic material. This increase, however, did not achieve statistical significance. On the contrary, there was a statistical correlation between iNOS (inducible NOS) and the severity of osteolysis around the prosthetic implant. Pharmacological control of the biosynthesis of NO may be considered in the prevention or treatment of loosening.

Effects of plasma treated PET and PTFE on expression of adhesion molecules by human endothelial cells in vitro

The aim of this study was to evaluate the expression of adhesion molecules on the surface of human endothelial cells in response to the systematic variation in materials properties by the ammonia plasma modification of polyethylene terephthalate (PET) and polytetrafluorethylene (PTFE). These adhesion molecules act as mediators of cell adhesion, play a role in the modulation of cell adhesion on biomaterials and therefore condition the response of tissues to implants. First and second passage human umbilical vein endothelial cells (HUVECs) were cultured on plasma treated and untreated PET and PTFE. HUVECs grown on polystyrene tissue culture coverslips and HUVECs stimulated with tumour necrosis factor (TNF- α) were used as controls. After 1 day and 7 days, the expression of adhesion molecules platelet endothelial cell adhesion molecule-1 (PECAM-1), intercellular adhesion molecule-1 (ICAM-1), Integrin α v β 3, vascular cell adhesion molecule-1 (VCAM-1), E-selectin, P-selectin and L-selectin were evaluated using flow cytometry and immunohistochemistry. There was a slight increase in positive cell numbers expressing the adhesion molecules ICAM-1 and VCAM-1 on plasma treated PET and PTFE. A significant increase in E-selectin positive cells on untreated PTFE was demonstrated after 7 days. Stimulation with TNF- α demonstrated a significant increase in the proportion of ICAM-1, VCAM-1 and E-selectin positive cells. Almost all cells expressed PECAM-1 and integrin α v β 3 on both materials and controls but did not express P- and L-selectin on any surface. When second passage cells were used, the expression of the adhesion molecules ICAM-1 and VCAM-1 was markedly increased on all surfaces but not with TNF- α. These significant differences were not observed in other adhesion molecules. These results were supported by immunohistochemical studies. The effects of plasma treated PET and PTFE on cell adhesion and proliferation was also studied. There was a 1.3-fold increase in cell numbers adhered on ammonia plasma treated PET compared to untreated PET and a 5.5-fold increase in cell numbers on treated PTFE compared to untreated PTFE after 1 day. This is significantly different when analysed statistically. After 7 days, cell number increased significantly on all surfaces compared to 1 day, except for untreated PTFE which conversely reduced by 41%. Cell number on the surface of untreated PET was no different to treated PET on days 1 and 7 when second passage cells were used. The study has shown that the plasma treatment of PET and PTFE with ammonia improves the adhesion and growth of endothelial cells and slightly upregulates the expression of adhesion molecules. This surface modification should promote colonisation of an artificial vascular prosthesis by endothelial cells and make it less vulnerable to immune system cells of the recipient. In addition, it should be considered which passage of cells is used due to the different adhesion features of different passages of HUVECs on untreated PET.

Short-Term Culturing Influences the Number of bcr/abl-Fused Cells Detected by Fluorescence in situ Hybridisation in Bone Marrow Aspirates of CML Patients

Fluorescence in situ hybridisation (FISH) has been proven as a helpful tool in diagnosis and monitoring of bcr/abl fusion in chronic myelogeneous leukaemia (CML). Since long-term cultures are known to decrease the number of bcr/abl-fused cells, it is questionable whether similar effects are detectable in short-term cultures, a technique often preceding FISH analysis. Therefore, we evaluated bone marrow aspirates of 10 CML patients at biopsy and after culturing for between 24 and 144 h by FISH. The percentage of bcr/abl-fused cells in FISH varied between 15 and 70% at biopsy. In samples with 15 and 30% of aberrant cells at biopsy, an increase of about 20% per day was seen within the first 48 h. In longer lasting cultures, the percentage of leukaemic cells then asymptotically approached a value of 60–70%. In patients with 38 and 50% of bcr/abl-fused cells at biopsy, an increase of about 20% could be detected in the first 24 h. Then 65–70% of the cells already bore the bcr/abl fusion, and the percentage of leukaemic cells was almost constant for longer lasting cultures up to 144 h. In patients with a percentage of about 70% before culturing, no increase in positive cells was detected. These results emphasize the impact of short-term culturing on the number of bcr/abl-fused cells. In particular, the importance for monitoring CML patients is obvious. Therefore the effect described should be taken into account in order to avoid misinterpretation and incorrect therapy decisions in CML patients.

THE EXTENT OF PERITUBULAR CD14 STAINING IN RENAL ALLOGRAFTS AS AN INDEPENDENT IMMUNOHISTOLOGICAL MARKER FOR ACUTE REJECTION

Previously, we demonstrated that in acute interstitial rejection, immunohistological staining of renal allograft biopsies with the CD14 mAb WT14, reacting with human monocytes/macrophages, shows a characteristic peritubular increase of positive cells. To test the diagnostic value of this CD14 positivity, we compared, in 154 unselected renal allograft biopsies, the extent of peritubular WT14 staining with (a) the original histological diagnosis, made with knowledge of clinical data, (b) the retrospectively and blindly scored histological diagnosis according to the criteria of the Banff classification, and (c) the eventual clinical diagnosis, which included evaluation of the response to therapy. The extent of peritubular WT14 positivity, blindly scored on cryostat sections of the frozen part of the biopsies, correlated positively with the probability of acute rejection (AR). When using a cutoff of 70% WT14 positivity for the diagnosis of AR, as extracted from a receiver operating characteristic curve, the WT14 diagnosis had a positive predictive value of 91% and a negative predictive value of 56%, compared with the original histological diagnosis. Compared with the Banff diagnosis of AR (grade I-III), these values were 95% and 47%, and compared with the clinical diagnosis, 84% and 63%, respectively. The WT14 diagnosis essentially corrected the original histological diagnosis in 7 cases, and was consistent with the eventual diagnosis in 5 equivocal cases. We conclude that the extent of peritubular CD14 positivity can be used as a marker for AR and can serve as a valuable additional criterion for AR in the histological examination of renal allograft biopsies.

Immunhistochemische Untersuchungen des mononukleären zellulären Infiltrates von papillären, follikulären und anaplastischen Schilddrüsenkarzinomen*

The mononuclear cellular infiltrate of thyroid neoplasms was examined by immunohistochemical techniques and compared to normal thyroid (control 1) and untreated Graves' disease tissue (control 2). Using a panel of monoclonal antibodies T-, B cells and macrophage antigens as well as the expression of activation-associated antigens and of MHC class II molecules were examined. In comparison to control 1 an increase of positive cells could be detected in carcinomas. This increase was significant for CD3+ and CD8+ T cells, for CD4+ macrophages and for HLA-DP+ cells in all types of carcinomas. In addition, in anaplastic carcinomas a significant increase of B cells, RM3/1+ macrophages, IL-2R+, ICAM-1+ and HLA-DQ+ cells was observed as well as of HLA-DR+ cells in follicular and of CD4+ T cells in papillary carcinomas. A comparison with control 2 revealed a significant increase of CD4+ cells (T cells and macrophages) in papillary and of CD8+ and RM3/1+ cells in anaplastic carcinomas. Our findings, an obvious increase of cell populations and of an upregulated expression of activation-associated antigens on these cells, indicated an immunological reaction caused by neoplastic thyrocytes. Further functional studies are however necessary.

Quantification and Distribution of Troponin-T in Cultures of Chick Embryo Myocardiocytes

We analyzed the distribution and labeling patterns of troponin-T, a protein involved in the regulation of striated muscle contraction, in myocardiocytes obtained from chick embryos in Hamburger and Hamilton's stage 25 and 39, and cultured for 8 days. Troponin-T expression was examined with indirect immunofluorescence, densitometry, fluorescence-activated cell sorting, electrophoresis and immunoblotting. The patterns of expression of troponin-T were compared with those of actin to determine possible correlations in different stages of chick embryo development and culture. Our findings show that in both stages of embryonic development, the cellular accumulation of troponin-T changed after 8 days of culture. Our results revealed a quantitative modification with time: after 4 days of culture there was a significant increase in this protein, followed by a slight additional increase after 8 days of culture. Flow cytometry findings confirmed these trends over time, showing a significant increase in positive cells after 4 days, followed by a smaller rise after 8 days of culture. In comparison with actin, this pattern was similar only in cells from Hamburger and Hamilton's stage 25 embryos.

Infection of rabbit T-cell and macrophage lines with human immunodeficiency virus.

We report the successful infection of two rabbit T-cell lines and one rabbit macrophage line with human immunodeficiency virus 1 (HIV-1). One T-cell line was a herpesvirus ateles transformant, the other T-cell line was a human T-cell leukemia virus I transformant, and the macrophage line was a simian virus 40 transformant. After infection with a high-titered HIV-1 stock, the rabbit cultures exhibited properties that closely mimic those of HIV-1-infected human cells. Productive infection was evident in cultures 7-14 days after infection, as shown by an increase in reverse transcriptase activity, a concomitant increase in positive cells detected by indirect immunofluorescence using serum from a patient with acquired immunodeficiency syndrome, and a decrease in cell viability. RNA gel blot hybridization and protein immunoblot analyses of infected cells indicated that all predicted viral transcripts and proteins were synthesized during the course of the infection. Proof that cell-free culture supernatants of the infected rabbit cell lines contained infectious virus was given by successful passage onto a susceptible human T-cell line. The ability of HIV-1 to infect transformed rabbit cell lines in vitro suggests that, with appropriate manipulation, the rabbit may provide a model for infection with HIV-1.

Differential induction of Epstein‐Barr virus‐related antigens in heterokaryon cultures

Different paterns of induction of Epstein-Barr virus (EBV)-related antigens were observed in heterokaryons produced by Sendai virus-mediated fusion of producer and non-producer human lymphoblastoid cells with various other cell types. EBV-related early antigens (EA) and viral capsid antigen (VCA) could obviously be induced in heterokaryons between producer cells (P3HR-1 and QIMR-WIL), normally expressing these natigens at very low frequency, and human FL or HeLa cells. Positive cells were detected as early as 3 h after fusion and there often followed a rapid increase in positive cells. In contrast, in heterokaryons between non-producer cells (Raji and NC-37) and FL or HeLa cells, only EA but not VCA was induced. EA induction was also evident in fusion of human lymphoblastoid cells with monkey cells (Vero) but with mouse cells (L-M(TK-) C11D and MCB-2) no EBV induction occurred. The EBV induction in heterokaryons was significantly enhanced by 5-iododeoxyuridine treatment.