Hybrid capture-based genomic profiling of circulating tumor DNA from patients with estrogen receptor-positive metastatic breast cancer.

BACKGROUND: Genomic changes that occur in breast cancer during the course of disease have been informed by sequencing of primary and metastatic tumor tissue. For patients with relapsed and metastatic disease, evolution of the breast cancer genome highlights the importance of using a recent sample for genomic profiling to guide clinical decision-making. Obtaining a metastatic tissue biopsy can be challenging, and analysis of circulating tumor DNA (ctDNA) from blood may provide a minimally invasive alternative. PATIENTS AND METHODS: Hybrid capture-based genomic profiling was carried out on ctDNA from 254 female patients with estrogen receptor-positive breast cancer. Peripheral blood samples were submitted by clinicians in the course of routine clinical care between May 2016 and March 2017. Sequencing of 62 genes was carried out to a median unique coverage depth of 7503×. Genomic alterations (GAs) in ctDNA were evaluated and compared with matched tissue samples and genomic datasets of tissue from breast cancer. RESULTS: At least 1 GA was reported in 78% of samples. Frequently altered genes were TP53 (38%), ESR1 (31%) and PIK3CA (31%). Temporally matched ctDNA and tissue samples were available for 14 patients; 89% of mutations detected in tissue were also detected in ctDNA. Diverse ESR1 GAs including mutation, rearrangement and amplification, were observed. Multiple concurrent ESR1 GAs were observed in 40% of ESR1-altered cases, suggesting polyclonal origin; ESR1 compound mutations were also observed in two cases. ESR1-altered cases harbored co-occurring GAs in PIK3CA (35%), FGFR1 (16%), ERBB2 (8%), BRCA1/2 (5%), and AKT1 (4%). CONCLUSIONS: GAs relevant to relapsed/metastatic breast cancer management were identified, including diverse ESR1 GAs. Genomic profiling of ctDNA demonstrated sensitive detection of mutations found in tissue. Detection of amplifications was associated with ctDNA fraction. Genomic profiling of ctDNA may provide a complementary and possibly alternative approach to tissue-based genomic testing for patients with estrogen receptor-positive metastatic breast cancer.

The role of multidetector computed tomography in predicting the site of gastroduodenal perforation.

OBJECTIVE: This study aims to evaluate the value of multidetector computed tomography (MDCT) in detecting the location of gastroduodenal perforation. PATIENTS AND METHODS: This cross-sectional descriptive study was conducted with 47 patients who underwent contrast-enhancing MDCT and were diagnosed with gastroduodenal perforation during surgery between July 2021 and June 2022. Radiologic findings included pneumoperitoneum (distribution and quantity) and analyzed the image findings for localizing the site of gastroduodenal perforation. RESULTS: Pneumoperitoneum was the most common finding [95.74% (45 out of 47 patients)]. Regarding air distribution, the sensitivity (Se) and negative predictive value (NPV) of abdominal free air and supramesocolic free air were the highest (100% for both). The accuracy (Acc) of supramesocolic free air was the highest (93.6%), followed by abdominal free air (89.4%). Subphrenic free air also had a high Acc value (89.4%), with Se, specificity (Sp), and positive predictive value (PPV) being 90%, 85,7%, and 97.3%, respectively. The Sp PPV of falciform ligament/ligamentum teres sign, and periportal free air were also high (100% for both). In contrast, retroperitoneal free air was valuable in determining retroperitoneal duodenal perforation with an Sp, Se of 100%, and Acc of 89.4%. The thickness of abdominal free air was ≥5.5 mm, suggesting gastroduodenal perforation with a Se, Sp, PPV, NPV, and Acc of 82.5%, 100%, 100%, 50%, and 85.1%, respectively. CONCLUSIONS: Subphrenic free air, periportal free air, falciform ligament sign, and the air above transverse mesocolon were correlated to gastric and duodenal bulb perforation. Retroperitoneal air indicates the perforation at the retroperitoneal duodenum. The thickness of abdominal free air ≥5.5 mm indicates gastric and duodenal bulb perforation.

Demonstration of polarization pulling using a fiber-optic parametric amplifier.

We report the observation of all-optical polarization pulling of an initially polarization-scrambled signal using parametric amplification in a highly nonlinear optical fiber. Broadband polarization pulling has been achieved both for the signal and idler waves with up to 25 dB gain using the strong polarization sensitivity of parametric amplifiers. We further derive the probability distribution function for the final polarization state, assuming a randomly polarized initial state, and we show that it agrees well with the experiments.

Diagnostic performance of quantitative signal intensity measurements on magnetic resonance imaging for distinguishing cerebellopontine angle meningioma from acoustic schwannoma.

OBJECTIVE: Our study investigated magnetic resonance imaging measurements for differentiating cerebellopontine angle (CPA) meningioma from vestibular schwannoma (VS). PATIENTS AND METHODS: This retrospective study compared 36 meningioma and 36 VS patients. The tumor volume (Vtumor) and peritumor edema index (EI) relationship was analyzed. T2-weighted three-dimensional gradient-echo image signal intensity (T23D) and apparent diffusion coefficient (ADC) differentiation cutoff values were defined. Mann-Whitney U test, independent-samples t-test, receiver operating characteristic curve, and Spearman's correlation analyses were applied. RESULTS: Meningioma had higher Vtumor (p=0.009) and EI (p=0.031) values than VS. Meningioma had significantly (p<0.001) lower values than VS for mean ADC (ADCmean: 0.841±0.083×10-3 vs.1.173±0.190×10-3 mm2/s), minimum ADC (ADCmin: 0.716±0.078×10-3 vs.1.045±0.178×10-3 mm2/s), tumor:white matter ADC ratio (rADC: 1.198±0.19 vs. 1.59±0.30), mean T23D (T23Dmean: 142.91±19.9 vs. 218.72±84.73), and tumor:adipose T23D ratio (rT23d: 0.19±0.06 vs. 0.30±0.28) Cutoff, sensitivity (Se), and specificity (Sp) values were ADCmin, 0.856×10-3 mm2/s (Se: 96.6%, Sp: 100%); ADCmean, 0.963×10-3 mm2/s (Se: 96.6%, Sp: 95.5%); rADC, 1.3189 (Se: 93.1%, Sp: 81.8%), T23Dmean (Se: 96.6%, Sp: 100%); rT23D, 0.1951 (Se: 89.7%, Sp: 100%), Vtumor, 14828.65 mm3 (Se: 75.0%, Sp: 66.7%), and EI, 1.1025 (Se: 47.2%, Sp: 100%). CONCLUSIONS: ADCmin, ADCmean, rADC, T23Dmean, rT23D, Vtumor, and EI, effectively discriminated meningioma from VS.

Discrimination between glioblastoma and solitary brain metastasis: a quantitative analysis based on FLAIR signal intensity.

OBJECTIVE: This study determined the diagnostic performance of fluid-attenuated inversion recovery (FLAIR) signal intensity (SI) in discriminating between glioblastoma (GBM) and solitary brain metastasis (SBM). PATIENTS AND METHODS: We recruited 40 patients with a histologically confirmed diagnosis of GBM or SBM who underwent conventional 3 Tesla magnetic resonance imaging before surgery or biopsy between August 2020 and January 2022. Three regions of interest were placed to assess FLAIR SI: the enhancing region (eFLAIR), the peritumoral region (pFLAIR), and the contralateral normal white matter (nFLAIR). The diagnostic performance of significantly different parameters between the two tumor entities was analyzed by receiver operating characteristic (ROC) curve analysis. RESULTS: The pFLAIR SI was significantly lower in GBM than in SBM (p < 0.05). The eFLAIR SI and the SI ratio eFLAIR and nFLAIR (e/nFLAIR) were significantly higher in GBM than in SBM (p < 0.05). On ROC curve analysis, the e/nFLAIR ratio provided the highest area under the curve value of 81%, with a sensitivity of 80.8% and a specificity of 85.7%, for distinguishing between the two tumor types. CONCLUSIONS: The eFLAIR, pFLAIR, and e/nFLAIR parameters are useful for differentiating between GBM and SBM.

Hospitalization of paediatric patients for methicillin-resistant Staphylococcus aureus skin and soft-tissue infection, 1998-2006.

Hospital discharge reports have provided data for studies of methicillin-resistant Staphylococcus aureus (MRSA) skin and soft-tissue infection (SSTI) studies. This analysis determined the sensitivity and positive predictive value of International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) code combinations to calculate hospitalization incidence rates, representativeness of a set of three ICD-9-CM codes to define MRSA SSTI, and hospitalization incidence rate trends for paediatric MRSA SSTIs in Los Angeles County (LAC). Using 133 cases from 31 hospitals, we found that the set of three ICD-9-CM codes used to define laboratory-confirmed cases had one of the highest positive predictive values (49%). There was no difference in age and race between those categorized using three codes vs. other code combinations. A dramatic increase in paediatric MRSA SSTI cases occurred in LAC during 1998-2006. We conclude that this combination of codes may be used to determine the rise of MRSA SSTIs in paediatric populations.

Novel urchin-like FeCo oxide nanostructures supported carbon spheres as a highly sensitive sensor for hydrazine sensing application.

Herein, we successfully fabricated a novel nanostructure based on hierarchical urchin-like FeCo oxide supported carbon spheres (FeCo Oxide/CSs) via a two-step hydrothermal method followed by a simple annealing step at 300 °C under air. It was found that such urchin-like FeCo Oxide/CSs structure exhibited superior catalytic activity towards hydrazine oxidation to CSs, Fe Oxide/CSs, Co Oxide/CSs, and FeCo Hydroxide/CSs material. In this regard, the FeCo Oxide/CSs displayed a wide linear detection range of 0.1-516.6 µM, low detection limit of 0.1 µM, and long-term stability. The material also showed good selectivity towards hydrazine detection in the presence of various interferences, such as uric acid, ascorbic acid, urea, dopamine, Na+, SO42-, K+, and Cl-. The excellent sensing performance of the FeCo Oxide/CSs was assumed to the unique hierarchical urchin structure with the high density and uniformity of nano-sized FeCo Oxide nanoneedles, which produced massive electroactive sites and enhanced charge transfer ability. The achieved results implied that the FeCo Oxide/CSs may be a great candidate for sensitive hydrazine detection.

Dynamic transcriptional regulatory complexes including BORIS, CTCF and Sp1 modulate NY-ESO-1 expression in lung cancer cells.

Previously, we reported that the paralogous zinc-finger proteins--CTCF and brother of the regulator of imprinted sites (BORIS), directly contribute to transcriptional regulation of NY-ESO-1 in lung cancer cells. To further examine mechanisms that mediate expression of this cancer-testis gene, we performed software-guided analysis of the NY-ESO-1 promoter region, which revealed several potential Sp1-binding motifs. Sequential 5-aza-2'deoxycytidine/depsipeptide FK228 treatment markedly induced BORIS expression and enhanced nuclear translocation of Sp1 in lung cancer cells. Transient transfection assays using promoter-reporter constructs, as well as gel-shift and chromatin immunoprecipitation experiments revealed that NY-ESO-1 promoter activity coincided with occupancy of the proximal Sp1-binding site in lung cancer cells. Mutations within the Sp1 recognition sequence specifically eliminated binding of Sp1 to this motif in vitro, and markedly diminished NY-ESO-1 promoter activity in vivo. siRNA-mediated inhibition of Sp1 expression decreased NY-ESO-1 promoter activity, whereas knock down of CTCF expression augmented NY-ESO-1 transcription in lung cancer cells. Co-immunoprecipitation experiments indicated that Sp1 physically interacts with BORIS but not with CTCF in vivo. Collectively, these findings suggest that BORIS recruits Sp1 to mediate de-repression of NY-ESO-1 during pulmonary carcinogenesis.

Inhibition of WNT signaling attenuates self-renewal of SHH-subgroup medulloblastoma.

The SMOOTHENED inhibitor vismodegib is FDA approved for advanced basal cell carcinoma (BCC), and shows promise in clinical trials for SONIC HEDGEHOG (SHH)-subgroup medulloblastoma (MB) patients. Clinical experience with BCC patients shows that continuous exposure to vismodegib is necessary to prevent tumor recurrence, suggesting the existence of a vismodegib-resistant reservoir of tumor-propagating cells. We isolated such tumor-propagating cells from a mouse model of SHH-subgroup MB and grew them as sphere cultures. These cultures were enriched for the MB progenitor marker SOX2 and formed tumors in vivo. Moreover, while their ability to self-renew was resistant to SHH inhibitors, as has been previously suggested, this self-renewal was instead WNT-dependent. We show here that loss of Trp53 activates canonical WNT signaling in these SOX2-enriched cultures. Importantly, a small molecule WNT inhibitor was able to reduce the propagation and growth of SHH-subgroup MB in vivo, in an on-target manner, leading to increased survival. Our results imply that the tumor-propagating cells driving the growth of bulk SHH-dependent MB are themselves WNT dependent. Further, our data suggest combination therapy with WNT and SHH inhibitors as a therapeutic strategy in patients with SHH-subgroup MB, in order to decrease the tumor recurrence commonly observed in patients treated with vismodegib.

Adenovirus-mediated p53 gene transfer in advanced non-small-cell lung cancer.

BACKGROUND: Preclinical studies in animal models have demonstrated tumor regression following intratumoral administration of an adenovirus vector containing wild-type p53 complementary DNA (Ad-p53). Therefore, in a phase I clinical trial, we administered Ad-p53 to 28 patients with non-small-cell lung cancer (NSCLC) whose cancers had progressed on conventional treatments. METHODS: Patients received up to six, monthly intratumoral injections of Ad-p53 by use of computed tomography-guided percutaneous fine-needle injection (23 patients) or bronchoscopy (five patients). The doses ranged from 10(6) plaque-forming units (PFU) to 10(11) PFU. RESULTS: Polymerase chain reaction (PCR) analysis showed the presence of adenovirus vector DNA in 18 (86%) of 21 patients with evaluable posttreatment biopsy specimens; vector-specific p53 messenger RNA was detected by means of reverse transcription-PCR analysis in 12 (46%) of 26 patients. Apoptosis (programmed cell death) was demonstrated by increased terminal deoxynucleotide transferase-mediated biotin uridine triphosphate nick-end labeling (TUNEL) staining in posttreatment biopsy specimens from 11 patients. Vector-related toxicity was minimal (National Cancer Institute's Common Toxicity Criteria: grade 3 = one patient; grade 4 = no patients) in 84 courses of treatment, despite repeated injections (up to six) in 23 patients. Therapeutic activity in 25 evaluable patients included partial responses in two patients (8%) and disease stabilization (range, 2-14 months) in 16 patients (64%); the remaining seven patients (28%) exhibited disease progression. CONCLUSIONS: Repeated intratumoral injections of Ad-p53 appear to be well tolerated, result in transgene expression of wild-type p53, and seem to mediate antitumor activity in a subset of patients with advanced NSCLC.

Antisense to SV40 early gene region induces growth arrest and apoptosis in T-antigen-positive human pleural mesothelioma cells.

Although SV40 oncoproteins have been detected in malignant pleural mesotheliomas (MPMs), their role in the pathogenesis and clinical behavior of these neoplasms remains controversial. In the present study, we sought to define the relevance of SV40 T/t antigen expression in established human mesothelioma cell lines deficient for p16INK4a as well as ARF expression. SV40 early region sequences were readily detected in genomic DNA isolated from pleural mesothelioma lines; however, levels of SV40 T/t antigen expression were highly variable in these cells. An adenoviral vector expressing an antisense transcript to SV40 early region inhibited T antigen expression and mediated significant growth inhibition and apoptosis in T-antigen-positive mesothelioma cells and SV40-transformed COS-7 cells. Abrogation of T/t antigen expression coincided with enhanced p21/WAF-1 expression, suggesting that restoration of p53-mediated pathways may have contributed to the growth inhibition and apoptosis induced by the antisense construct. These effects were not observed after similar treatment of mesothelioma or lung cancer cells containing no SV40 DNA sequences. Collectively, these data suggest that SV40 oncoproteins contribute to the malignant phenotype of pleural mesotheliomas and indicate that interventions designed to abrogate their expression may be efficacious in the treatment of individuals with these neoplasms.

Coherently Tunable Triangular Trefoil Phaseonium Metamaterial.

Phaseonium is a three-level Λ quantum system, in which a coherent microwave and an optical control (pump) beams can be used to actively modulate the dielectric response. Here we propose a new metamaterial structure comprising of a periodic array of triangular phaseonium metamolecules arranged as a trefoil. We present a computational study of the spatial distribution of magnetic and electric fields of the probe light and the corresponding transmission and reflection, for various parameters of the optical and microwave beams. For specific values of the probing frequencies and control fields, the phaseonium can display either metallic or dielectric optical response. We find that, in the metallic regime, the phaseonium metamaterial structure supports extremely large transmission, with optical amplification at large enough intensity of the microwave thanks to strong surface plasmon coupling; while, in the dielectric regime without microwave excitation, the transmission bandwidth can be tuned by varying the control beam intensity. Implementation of such phaseonium metamaterial structure in solid-state systems, such as patterned crystals doped with rare-earth elements or dielectric matrices embedded with quantum dots, could enable a new class of actively tunable quantum metamaterials.

Effect of organoclay on morphology and properties of linear low density polyethylene and Vietnamese cassava starch biobased blend.

Linear low density polyethylene (LLDPE)/thermal plastic starch (TPS) blend was studied to prepare the biobased nanocomposite material using organoclay nanofil15 (N15) modified by alkilammonium as the reinforced phase. The LLDPE/TPS blend and its nanocomposites were elaborated by melt mixing method at 160 °C for 7 min. And the compounded sample was filmed by blowing method at three different zones of temperature profile which are 160-170-165 °C. The good dispersion of clay in the polymer blend matrix is showed by X-ray diffraction (XRD) and transmission electronic microscopy (TEM), and a semi-exfoliated structure was obtained. The thermal and mechanical properties of materials are enhanced when N15 is added to the mixture. The effect of N15 on morphology and particles size of TPS phase is also investigated. The biodegradation test shows that more than 60% in weight of LLDPE/TPS film is degraded into CO2, H2O, methane and biomass after 5 months in compost soil.

Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer.

PURPOSE: To determine the safety and tolerability of adenovirus-mediated p53 (Adp53) gene transfer in sequence with cisplatin when given by intratumor injection in patients with non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients with advanced NSCLC and abnormal p53 function were enrolled onto cohorts receiving escalating dose levels of Adp53 (1 x 10(6) to 1 x 10(11) plaque-forming units [PFU]). Patients were administered intravenous cisplatin 80 mg/m(2) on day 1 and study vector on day 4 for a total of up to six courses (28 days per course). Apoptosis was determined by the terminal deoxynucleotidyl- transferase-dUTP nick-end labeling assay. Evidence of vector-specific sequences were determined using reverse-transcriptase polymerase chain reaction. Vector dissemination and biodistribution was monitored using a series of assays (cytopathic effects assay, Ad5 hexon enzyme-linked immunosorbent assay, vector-specific polymerase chain reaction assay, and antibody response assay). RESULTS: Twenty-four patients (median age, 64 years) received a total of 83 intratumor injections with Adp53. The maximum dose administered was 1 x 10(11) PFU per dose. Transient fever related to Adp53 injection developed in eight of 24 patients. Seventeen patients achieved a best clinical response of stable disease, two patients achieved a partial response, four patients had progressive disease, and one patient was not assessable. A mean apoptotic index between baseline and follow-up measurements increased from 0.010 to 0.044 (P =.011). Intratumor transgene mRNA was identified in 43% of assessable patients. CONCLUSION: Intratumoral injection with Adp53 in combination with cisplatin is well tolerated, and there is evidence of clinical activity.

Isolation and physical characterization of random insertions in Staphylococcal nuclease.

Using genetic engineering techniques we generated randomly located internal tandem duplications of random size within Staphylococcal nuclease. Those insertions, possessing greater than 0.1% of normal activity, were sequenced and characterized physically. Insertions were found to begin and end in regions possessing secondary structure as well as in regions without secondary structure. All proteins remained folded and monomeric, although one mutant appeared, by both circular dichroism and size exclusion chromatography, to be partially unfolded. The stability of the insertions as assayed by guanidine hydrochloride denaturation ranged from nearly normal to destabilized by almost 4 kcal per mol. The activities of the insertion mutants ranged from 1/30 to 1/2000 of the parental nuclease.

MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways.

Oncogenic RAS promotes production of reactive oxygen species (ROS), which mediate pro-malignant signaling but can also trigger DNA damage-induced tumor suppression. Thus RAS-driven tumor cells require redox-protective mechanisms to mitigate the damaging aspects of ROS. Here, we show that MutT Homolog 1 (MTH1), the mammalian 8-oxodGTPase that sanitizes oxidative damage in the nucleotide pool, is important for maintaining several KRAS-driven pro-malignant traits in a nonsmall cell lung carcinoma (NSCLC) model. MTH1 suppression in KRAS-mutant NSCLC cells impairs proliferation and xenograft tumor formation. Furthermore, MTH1 levels modulate KRAS-induced transformation of immortalized lung epithelial cells. MTH1 expression is upregulated by oncogenic KRAS and correlates positively with high KRAS levels in NSCLC human tumors. At a molecular level, in p53-competent KRAS-mutant cells, MTH1 loss provokes DNA damage and induction of oncogene-induced senescence. In p53-nonfunctional KRAS-mutant cells, MTH1 suppression does not produce DNA damage but reduces proliferation and leads to an adaptive decrease in KRAS expression levels. Thus, MTH1 not only enables evasion of oxidative DNA damage and its consequences, but can also function as a molecular rheostat for maintaining oncogene expression at optimal levels. Accordingly, our results indicate MTH1 is a novel and critical component of oncogenic KRAS-associated malignancy and its inhibition is likely to yield significant tumor-suppressive outcomes in KRAS-driven tumors.

Gene delivery into malignant cells in vivo by a conjugated adenovirus/DNA complex.

Current viral delivery systems suffer from disadvantages that may limit the rate at which therapeutic gene expressing constructs can be tested both in vitro and in vivo. In this study, our focus was to develop a simple gene delivery system for the rapid and reproducible testing of therapeutic genes in cancer cells both in vitro and in vivo. We report here that a delivery system based on using a conjugated adenovirus in complex from with a DNA plasmid can be used for not only delivering genes in vitro but also for efficient and reproducible delivery in vivo. Replication defective adenoviral particles were chemically modified by covalent attachment of poly-L-lysine (PLL) to the viral capsid, allowing for direct interaction with DNA. The adenovirus/PLL conjugate (Adv/PLL) was used to deliver the plasmid pCMV/beta-gal to several different cancer cell lines (i.e., lung, cervical) in vitro and resulted in transduction efficiencies as high as 52% as determined by histochemical staining. On direct intralesional injection of the Adv/PLL/DNA complex into subcutaneous tumors, transduction efficiencies greater than 35% could also be achieved. As a result, this system provides a simple method for delivering and testing therapeutic genes in cells both in vitro and in vivo, prior to the further development of gene therapy vectors for both malignant and benign disease.

Delivery of the p53 tumor suppressor gene into lung cancer cells by an adenovirus/DNA complex.

An adenovirus/DNA complex was constructed by chemically linking poly-L-lysine to the capsid of the replication-defective adenovirus dl312, allowing for coupling with plasmid DNA by an ionic interaction. We have previously demonstrated that this adenovirus/DNA complex can efficiently transduce malignant cells with a plasmid expressing the beta-galactosidase gene both in vitro and in vivo. In this report, we show that this system can deliver a therapeutic gene that encodes for the tumor suppressor protein p53 to lung cancer cells, both in vitro and in vivo, leading to significant biological effects. Transfection of the p53-negative human lung cancer cell line H1299 with the adenovirus/DNA complex carrying a plasmid expressing the p53 gene resulted in high levels of p53 protein and induction of apoptosis. Injection of the complex carrying the p53 gene to subcutaneous tumor sites 5 days after tumor cell implantation resulted in a significant inhibition of tumorigenicity as measured by the number and size of tumors that developed 21 days after treatment. Three and six injections of the complex carrying the p53 gene into H1299 subcutaneous tumor nodules led to significant dose-related tumor growth suppression 18 days after the first injection compared with control-treated tumors. This adenovirus/DNA complex, therefore, is capable of efficiently delivering the p53 gene into malignant cells in vitro and in vivo and now provides a general gene delivery vector that is simple to construct and capable of testing therapeutic genes in malignant cells.

Lectin-dependent cell-mediated cytotoxicity and natural killer function in rejecting and infected lung allografts.

Differentiation between rejection and infection of lung allografts remains difficult. The effects of these two pathologic entities on the cytolytic activity of bronchoalveolar lavage (BAL) and PBL were investigated. Left lung allotransplantation was performed on 16 mongrel dogs of which 12 were available for complete studies. All animals received CA, AZA, and PRED for 2 weeks. Four grafts developed left lower lobe Gram negative pneumonia. The eight remaining recipients progressed gradually to severe rejection after acute reduction of immunosuppression. Cytolytic activity of blood and left lung BAL lymphocytes was quantitated by the natural killer (NK) and lectin-dependent cell-mediated cytotoxicity (LDCMC) assays. Two additional groups serving as controls were either given a 10-day course of immunosuppressants or had right lower lobe pneumonia induced by transbronchial inoculation of gram negative bacteria. Immunosuppressed control animals showed significant depression of PBL and BAL lymphocyte LDCMC and NK activity. Similarly, BAL lymphocytes expressed very low LDCMC in normal allografts (2.8 +/- 0.8%). Once rejection developed and progressed, LDCMC became significantly higher (15.6 +/- 2.2 and 52.7 +/- 2.8% in mild and severe rejection, respectively). There was no detectable NK activity in rejecting lung allografts. BAL lymphocytes from infected allografts, on the other hand, showed an elevation of both NK and LDCMC activity (9.1 +/- 1.1 and 14.6 +/- 1.0%, respectively). Similarly, bacterial pneumonia in control animals manifested an increase in NK and LDCMC activity in lung and blood. PBL lymphocytes of lung allograft recipients, however, had increased NK and LDCMC activity in both rejection and infection. LDCMC/NK activity ratio (LM/NK index) of lung lymphocytes was significantly higher in rejecting allografts (11.2 +/- 1.0 and 12.4 +/- 1.6 for mild and severe rejection, respectively) than in infected ones (1.2 +/- 0.3, P < 0.0001). It appears, from this study, that rejection of the lung allograft results in alterations in BAL lymphocyte phenotypes and functions that differ from those associated with bacterial infection. Such differences may be useful in distinguishing episodes of acute allograft rejection from bacterial infection.

Inhibition of natural killer cell-mediated lysis of tumor cells by normal and regenerating bone marrow.

The natural killer (NK) cells which can lyse certain tumor cells during brief incubation in vitro have also been postulated to be the cells responsible for natural resistance to transplanted hemopoietic cells in vivo. To test this hypothesis, we have now measured: 1) the ability of bone marrow cells to compete with tumor cells as targets for spleen NK cells and 2) the effect of a brief incubation with spleen cells on the hemopoietic grafting potential of bone marrow cells. Firstly, when CBA/J mouse spleen cells were incubated with 51Cr-labelled YAC tumor cells together with DBA/2 mouse bone marrow cells, tumor cell lysis was reduced compared with incubation of spleen cells with tumor cells alone. Tumor cell lysis was even less when post-irradiation regenerating bone marrow was used. Secondly, C57B1/6 mouse bone marrow cells incubated with an excess of DBA/2 mouse spleen cells showed a reduced ability to produce hemopoietic spleen colonies in irradiated 129/J mice, whereas incubation with either thymus cells or fewer spleen cells produced no such effect. The results show that, when incubated with spleen cells under the conditions of a standard NK cell assay, regenerating bone marrow cells competitively inhibit the killing of YAC tumor cells and bone marrow progenitor cells are rendered ineffective in their hemopoietic colony-forming potential (CFU-s). These findings suggest that certain hemopoietic progenitor cells and YAC tumor cells can both serve as targets for NK cells, consistent with the view that the spontaneous cytolysis of tumor cells in vitro and natural resistance to bone marrow transplantation in vivo are mediated by cells of a common lineage.