Hamster Lung Tissue Research Articles

Alisol B 23-acetate broadly inhibits coronavirus through blocking virus entry and suppresses proinflammatory T cells responses for the treatment of COVID-19

IntroductionEmerging severe acute respiratory syndrome (SARS) coronavirus (CoV)-2 causes a global health disaster and pandemic. Seeking effective anti-pan-CoVs drugs benefit critical illness patients of coronavirus disease 2019 (COVID-19) but also may play a role in emerging CoVs of the future. ObjectivesThis study tested the hypothesis that alisol B 23-acetate could be a viral entry inhibitor and would have proinflammatory inhibition for COVID-19 treatment. MethodsSARS-CoV-2 and its variants infected several cell lines were applied to evaluate the anti-CoVs activities of alisol B 23-aceate in vitro. The effects of alisol B 23-acetate on in vivo models were assessed by using SARS-CoV-2 and its variants challenged hamster and human angiotensin-converting enzyme 2 (ACE2) transgenic mice. The target of alisol B 23-acetate to ACE2 was analyzed using hydrogen/deuterium exchange (HDX) mass spectrometry (MS). ResultsAlisol B 23-acetate had inhibitory effects on different species of coronavirus. By using HDX-MS, we found that alisol B 23-acetate had inhibition potency toward ACE2. In vivo experiments showed that alisol B 23-acetate treatment remarkably decreased viral copy, reduced CD4+ T lymphocytes and CD11b+ macrophages infiltration and ameliorated lung damages in the hamster model. In Omicron variant infected human ACE2 transgenic mice, alisol B 23-acetate effectively alleviated viral load in nasal turbinate and reduced proinflammatory cytokines interleukin 17 (IL17) and interferon γ (IFNγ) in peripheral blood. The prophylactic treatment of alisol B 23-acetate by intranasal administration significantly attenuated Omicron viral load in the hamster lung tissues. Moreover, alisol B 23-acetate treatment remarkably inhibited proinflammatory responses through mitigating the secretions of IFNγ and IL17 in the cultured human and mice lymphocytes in vitro. ConclusionAlisol B 23-acetate could be a promising therapeutic agent for COVID-19 treatment and its underlying mechanisms might be attributed to viral entry inhibition and anti-inflammatory activities.

Hamster organotypic modeling of SARS-CoV-2 lung and brainstem infection

SARS-CoV-2 has caused a global pandemic of COVID-19 since its emergence in December 2019. The infection causes a severe acute respiratory syndrome and may also spread to central nervous system leading to neurological sequelae. We have developed and characterized two new organotypic cultures from hamster brainstem and lung tissues that offer a unique opportunity to study the early steps of viral infection and screening antivirals. These models are not dedicated to investigate how the virus reaches the brain. However, they allow validating the early tropism of the virus in the lungs and demonstrating that SARS-CoV-2 could infect the brainstem and the cerebellum, mainly by targeting granular neurons. Viral infection induces specific interferon and innate immune responses with patterns specific to each organ, along with cell death by apoptosis, necroptosis, and pyroptosis. Overall, our data illustrate the potential of rapid modeling of complex tissue-level interactions during infection by a newly emerged virus.

Quantitative proteomics of hamster lung tissues infected with SARS-CoV-2 reveal host factors having implication in the disease pathogenesis and severity.

Syrian golden hamsters (Mesocricetus auratus) infected by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) manifests lung pathology. In this study, efforts were made to check the infectivity of a local SARS‐CoV‐2 isolate in a self‐limiting and non‐lethal hamster model and evaluate the differential expression of lung proteins during acute infection and convalescence. The findings of this study confirm the infectivity of this isolate in vivo. Analysis of clinical parameters and tissue samples show the pathophysiological manifestation of SARS‐CoV‐2 infection similar to that reported earlier in COVID‐19 patients and hamsters infected with other isolates. However, diffuse alveolar damage (DAD), a common histopathological feature of human COVID‐19 was only occasionally noticed. The lung‐associated pathological changes were very prominent on the 4th day post‐infection (dpi), mostly resolved by 14 dpi. Here, we carried out the quantitative proteomic analysis of the lung tissues from SARS‐CoV‐2‐infected hamsters on day 4 and day 14 post‐infection. This resulted in the identification of 1585 proteins of which 68 proteins were significantly altered between both the infected groups. Pathway analysis revealed complement and coagulation cascade, platelet activation, ferroptosis, and focal adhesion as the top enriched pathways. In addition, we also identified altered expression of two pulmonary surfactant‐associated proteins (Sftpd and Sftpb), known for their protective role in lung function. Together, these findings will aid in understanding the mechanism(s) involved in SARS‐CoV‐2 pathogenesis and progression of the disease.

Inactivation and Elimination of SARS-CoV-2 in Biosamples Using Simple Fixatives and Ultrafiltration

The Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) causes Coronavirus disease-2019 (COVID-19), which is an ongoing pandemic that has significantly affected the health, economy, and socio-economic status of individuals worldwide. Laboratory research using in vitro, ex vivo and in vivo models has been accelerated to understand the pathogenesis of SARS-CoV-2 infection. However, such experimental research involving SARS-CoV-2 is restricted to biocontainment/safety level-3 (BSL-3) settings, due to the high pathogenicity of this virus. Since many of the downstream analyses of SARS-CoV-2-infected biological samples need to be conducted in a non-BSL3 setting, it is important to ensure that the samples are fully decontaminated and safe for subsequent analysis. Here, we report the effectiveness of standard procedures used to fix cells and tissues for pathological analysis, including 2% or 4% paraformaldehyde, 50%–70% ethanol, 10% neutral buffered formalin and ultrafiltration using membranes with a molecular weight cut-off (MWCO) ranging from 3 to 30 kDa, for inactivating or eliminating SARS-CoV-2. We validated these methods in experimental laboratory samples, such as viral inoculum in cell culture media, SARS-CoV-2 infected host cells and animal tissue lysates. We found that 15 minutes’ treatment of viral inoculum (105 plaque-forming units; PFU) or SARS-CoV-2 infected cells with paraformaldehyde or 70% ethanol resulted in complete inactivation of the virus. The treatment of infected hamster lung tissues with 10% neutral buffered formalin also fully inactivated the virus. However, only 3 kDa ultracentrifuge filter was effective in eliminating the virus to an undetectable limit in the filtrate. Our validated methods are useful for decontaminating biological samples to reduce infection risk and safe handling in BSL2 facilities.

Expression Analyses of MicroRNAs in Hamster Lung Tissues Infected by SARS-CoV-2.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is an infectious disease with multiple severe symptoms, such as fever over 37.5°C, cough, dyspnea, and pneumonia. In our research, microRNAs (miRNAs) binding to the genome sequences of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory-related coronavirus (MERS-CoV), and SARS-CoV-2 were identified by bioinformatic tools. Five miRNAs (hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-195-5p, hsa-miR-16-5p, and hsa-miR-196a-1-3p) were found to commonly bind to SARS-CoV, MERS-CoV, and SARS-CoV-2. We also identified miRNAs that bind to receptor proteins, such as ACE2, ADAM17, and TMPRSS2, which are important for understanding the infection mechanism of SARS-CoV-2. The expression patterns of those miRNAs were examined in hamster lung samples infected by SARS-CoV-2. Five miRNAs (hsa-miR-15b-5p, hsa-miR-195-5p, hsa-miR-221-3p, hsa-miR-140-3p, and hsa-miR-422a) showed differential expression patterns in lung tissues before and after infection. Especially, hsa-miR-15b-5p and hsa-miR-195-5p showed a large difference in expression, indicating that they may potentially be diagnostic biomarkers for SARS-CoV-2 infection.

Establishment of fast-growing serum-free immortalised cells from Chinese hamster lung tissues for biopharmaceutical production

Chinese hamster (Cricetulus griseus) ovary-derived Chinese hamster ovary (CHO) cells are the most commonly used mammalian hosts for the industrial production of recombinant therapeutics because of their ability to fold, assemble, and perform post-translational modifications, such as glycosylation, on proteins. They are also valuable for their ability to grow in serum-free suspension cultures. In this study, we established a cell line derived from lung tissue of Chinese hamsters, named Chinese hamster lung (CHL)-YN cells. The biosafety of CHL-YN cells was confirmed by in vitro sterility testing, mycoplasma detection, and reverse transcriptase assays. One of the key characteristics of CHL-YN cells was their doubling time of 8.1 h in chemically defined culture medium; thus, they proliferate much faster than conventional CHO cells and general mammalian cells. Transgenes could be introduced into CHL-YN cells with high efficiency. Finally, between 50% to > 100% of the amount of glycosylated immunoglobulin G (IgG)1 produced by CHO-K1 cells was produced by CHL-YN cells over a shorter period of time. In summary, fast-growing CHL-YN cells are a unique cell line for producing recombinant proteins.

Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection

Leptospirosis causes severe clinical signs more frequently in men than in women, but the mechanism underlying the gender differences in leptospirosis remains unclear. In this study, petechial hemorrhage was observed in male but not in female hamster lung tissues infected with Leptospira interrogans serovar Hebdomadis at 120 h pi, demonstrating that male hamsters were more susceptible to the development of a severe disease upon Leptospira infection. No leptospiral DNA was detected in the lung tissues at 120 h pi when pulmonary hemorrhage was observed, indicating that pulmonary hemorrhage is attributable to the immune reactions of the host rather than from the direct effect of leptospires. The upregulation of nitric oxide synthase genes in the hamsters without pulmonary hemorrhage, inos and enos in female hamsters at 96 h pi and enos in male animals without hemorrhage at 120 h pi, may suggest that nitric oxide has a suppressive effect on leptospirosis-associated pulmonary hemorrhage.

2016 Lucian Award

Fifty years after it was established through a generous bequest to McGill University,1 the 2016 Louis and Artur Lucian Award for research in circulatory disease has been presented to Brian Kobilka, MD, of Stanford University. Kobilka is being honored for his enormous contributions to the understanding of G protein–coupled receptors (GPCRs), the versatile family of receptors that mediate the majority of cellular responses to hormones, neurotransmitters, and some types of sensory input. “Kobilka revealed the molecular configuration of the β2-adrenergic receptor (β2AR) through the use of crystallography, then doggedly pursued research in this area until he discovered how the receptor functions. He has now expanded his work to a whole range of GPCRs. Many of the other candidates for the 2016 Lucian award were extremely productive and highly cited, but his science was head and shoulders above the others. With a very long array of papers in high-impact and difficult-to-publish-in journals— Cell , Science , Nature , and PNAS —Kobilka’s contributions have been recognized by his peers as cutting-edge work that advances the field significantly, not as small incremental discoveries,” says James Martin, MD, chair of the McGill University Department of Medicine and new chair of the Lucian award committee. During his internship at Barnes Hospital, Kobilka often treated unstable patients with medications acting on GPCRs, including the adrenergic and muscarinic receptors regulating heart rate and blood pressure and opioid receptors controlling pain. His interest in intensive care medicine led Kobilka to apply for cardiology fellowships, and he was drawn to the program at Duke University because it encouraged fellows to pursue basic research. Kobilka joined the laboratory of Robert Lefkowitz, MD, where the β2AR had been identified and Jeff Benovic, a graduate student, had recently succeeded in purifying enough β2AR …

A new cell line derived from newborn Chinese hamster lung tissue.

A new cell line derived from newborn Chinese hamster lung tissue.

Evaluation of magnolia bark extract in chromosomal aberration assays

Magnolia bark extract (MBE) has been used historically in traditional Chinese and Japanese medicines, and more recently as a component of dietary supplements and cosmetic products. The genotoxic potential of MBE was studied in two in vitro chromosomal aberration assays. In Chinese hamster ovary (CHO) cells, exposure for 3 h to MBE at concentrations of 0–30 μg/ml in the absence of a metabolic activation system (S9) and 0–7 μg/ml with S9 did not induce chromosomal aberrations, whereas higher concentrations were cytotoxic and did not allow for analysis of aberrations. Extended exposure for 18 h without metabolic activation at concentrations up to 15 μg/ml also resulted in a negative response. In V79 cells derived from Chinese hamster lung tissue, treatment for 6 h with concentrations up to 52 and 59 μg/ml in the absence and presence of S9, respectively, did not increase the incidence of chromosomal aberrations compared to negative controls. Furthermore, MBE exposure for 24 h without metabolic activation did not induce aberrations. The results of these studies demonstrate that MBE is not genotoxic under the conditions of the in vitro chromosomal aberration assays in CHO and V79 cells, and support the safety of MBE.

Metabolism of the beta-oxidized intermediates of N-nitrosodi-n-propylamine: N-nitroso-beta-hydroxypropylpropylamine and N-nitroso-beta-oxopropylpropylamine.

The rat liver carcinogen N-nitrosodi-n-propylamine (NDPA) is metabolized to a propylating and methylating species in vivo. Metabolism to a methylating species is believed to require an initial hydroxylation by cytochrome P450s (P450s) to N-nitroso-beta-hydroxypropylpropylamine (NHPPA), which is oxidized to N-nitroso-beta-oxopropylpropylamine (NOPPA), followed by a P450-mediated depropylation to beta-oxopropyldiazotate, which non-enzymatically breaks down to the methylating agent. Purified rat liver P450 2B1 and rabbit liver 2E1 in the reconstituted system and liver microsomes from phenobarbital (PB) and pyridine (Pyr) treated rats readily metabolized NOPPA to a methylating species as determined by the in vitro formation of 7-methylguanine (m7Gua) in DNA. Exposure of cells derived from the human liver epithelium transfected with human 2E1 (T5-2E1) to NOPPA resulted in the formation of m7Gua DNA adducts and a dose dependent toxicity. In vitro incubation of NHPPA with microsomes from PB, Pyr and non-treated (NT) rats and a human microsomal sample also resulted in m7Gua formation. P450s 2B1 and 2E1 oxidized NHPPA to NOPPA, forming 16.5 +/- 3.1 and 20.0 +/- 4.4 pmol NOPPA/pmol P450 in 1 h, respectively. Rat liver cytosol, in the presence of NAD+, oxidized NHPPA to NOPPA at a rate of 13.7 +/- 3.0 pmol/min/mg protein while microsomes from NT rats catalyzed this reaction at 95.6 +/- 16.5 pmol/min/mg protein. Cells derived from hamster lung tissue (V79 control) and T5-neo cells oxidized NHPPA to NOPPA. This oxidation was about 15 fold higher in T5-2E1 or V79 cells transfected with human 2E1 or rat 2B1, respectively. The results are consistent with the putative sequential oxidation pathway and suggest that, at the concentrations tested, oxidation of NHPPA to NOPPA may be predominantly mediated by cytochrome P450s. In addition, it appears that rabbit, rat and human P450 2E1 can catalyze both oxidations.

Use of precision-cut tissue slices in organ culture to study metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by hamster lung, liver and kidney

The pharmacokinetics of in vitro metabolism of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; concentration range 0.03–250 μM) and its proximal metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL; 0.04–250 μM), were determined in Syrian golden hamster liver, lung, and kidney tissue slices in organ culture under identical experimental conditions. In the lung, a target organ for NNK animal carcinogenesis, total NNK metabolism was relatively low (maximum 23%) and oxidative metabolism by α-hydroxylation to DNA-reactive intermediates accounted for 13–31% of metabolism. The liver, a non-target organ for NNK carcinogenesis, showed the highest capacity to metabolise NNK (total metabolism 80%), and α-hydroxylation accounted for 12–25% of metabolism. The kidney, another non-target organ, also showed a low capacity for NNK metabolism (maximum 32%) and α-hydroxylation accounted for <3% of metabolism. Detoxification of NNK by pyridyl N-oxidation was similar in lung (5–22%) and liver (5–23%), and negligible in kidney (<2%), while carbonyl reduction of NNK to NNAL was greatest in the kidney (95–100%), followed by liver (59–79%) and lung (47–81%). NNAL is devoid of biological activity in the hamster and total metabolism was about tenfold lower than that of NNK in all tissues (<13% liver; <4% lung and kidney). In the liver, α-hydroxylation was the predominant pathway of NNAL metabolism at almost all concentrations (31–68% of total metabolism), whereas N-oxidation prevailed in the kidney (47–68%). In the lung, a concentration dependent decrease in the relative amount of α-hydroxylation (23–72%) with increasing NNAL concentrations occurred at the expense of N-oxidation (25–72%). Little or no metabolism of NNAL back to NNK was evident in any tissue.

Structural and functional characteristics of peripheral pulmonary parenchyma in golden hamsters

Mechanical properties of the peripheral pulmonary parenchyma of freshly excised hamster lung tissue were examined to evaluate determinants of displacement-tension relationships with regard to structural constituents of the alveolar wall. A tissue segment measuring 50 x 50 x 400-600 microns and consisting mostly of the alveolar wall was prepared from the lung parenchyma adjacent to the pleura. By use of a constant speed maneuver for extension and relaxation of this minute preparation, displacement-tension relationships of peripheral pulmonary parenchyma were examined in a bath filled with 37 degrees C physiological buffer solution. The specimen was repeatedly extended up to 20-40 mg, a little above a point resembling "yield" in displacement-tension relationships. Analyses of displacement-tension relationships constantly showed double exponential relations. The first component at the lower strain was approximated by sigma 1 = A1(e alpha 1 epsilon - 1) and the second component beyond the inflection (yield) point was sigma = s1 + s2 = A1(e alpha 1 epsilon - 1) + A2(e alpha 2 epsilon - 1), where sigma, A, alpha, and epsilon represent stress, constant determined by tissue quantity, elasticity constant, and strain, respectively. Immersion of the lung specimen into elastase resulted in decreases of only alpha 1, and collagenase reduced alpha 2 but not alpha 1. Hyaluronidase, acetylcholine, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and norepinephrine did not alter alpha 1 or alpha 2. These observations suggest that alpha 1 and alpha 2 of the peripheral pulmonary parenchyma are mechanical indexes of elastin and collagen characters, respectively.

Comparisons of type II cells isolated from rat, hamster, pig and human lung tissue

Comparisons of type II cells isolated from rat, hamster, pig and human lung tissue

Stress-strain characteristics of normal and emphysematous hamster lung strips

A simple mathematical model of the one dimensional, stress-strain behavior of hamster lung tissue based on strain energy considerations was tested in degassed, uniaxially stretched strips obtained from normal and emphysematous hamster lungs cycled in saline. The relationship between Eulerian stress (σ) and extension ratio (λ) was found to take the form σ = ( λ 2 − 1/ λ) × f( λ) was experimentally determined. Stress in six normal and five emphysematous strips was calculated by dividing the tension at each stretch increment by the strip cross-sectional area. Plotting σλ/( λ 2−1 versus a function of the form e nλ yielded a linear expression for f(λ), me nλ + b, where n = 2. The complete stress-strain behavior of hamster lung strip tissue could then be expressed as a simple function of λ over a range of λ = 1.0−2.0: σ=(λ 2−1/λ)( me 2λ b) The values of the constants m and b depend solely upon the mechanical properties of the elastic and collagen fiber networks in these atelectatic, saline cycled lung strips. The slope m = 0.151, and the intercept b = 0.416 in normal strips ( r = 0.98).In emphysematous strips m = 0.016 and b = −0.199 ( r = 0.82). Given the smaller m found for emphysematous strips, less strain energy accumulated with increasing stretch and did not even begin in these strips until λ = 1.3. Further, the fit of the equation to the data was not as good for emphysematous as for normal strips. We conclude that the above equation adequately describes the stress-strain properties of normal hamster lung strips tissue but is not as good in emphysematous strips where the disease is patchy.

Immunohistochemical and Biochemical Identification of Pepsinogen Isozymes in the Hamster Lungs: Induction by Polychlorinated Biphenyls

Pepsinogens are acid protease enzymes of pepsin usually found in gastric mucosa. In the present study, we demonstrated the presence of pepsinogen isozymes in male Syrian golden hamster lung tissues by a combined immunohistochemical and biochemical approach. Immunohistochemically, using rat pepsinogen 1 antibody, pepsinogen positive cells were observed mainly in the epithelia of the terminal bronchioles. They demonstrated morphological features of Clara cells. The pepsinogen isozyme pattern of lung tissue determined by polyacrylamide gel electrophoresis was similar to that of stomach mucosa. Treatment of hamsters with polychlorinated biphenyls at a dose of 500 mg/kg body weight ip caused a 2.8-fold increase in pepsinogen content (p less than 0.01) as well as increase in numbers of pepsinogen positive cells in the lung.

The effect of bleomycin on the uptake and incorporation of [14C] choline into phospholipids in hamster lung tissue slices

Intratracheal administration of the anticancer drug bleomycin to hamsters produced an increase in the uptake and incorporation of [14C]choline into phospholipids of lung slices in vitro. The stimulatory effect is opposite to the results obtained previously using [14C]acetate and would appear to occur distal to cytidine diphosphocholine. Although alternate explanations are possible, the results are consistent with morphological evidence, published by others, indicating an increase in lung phospholipid following bleomycin treatment, and illustrate the significance of precursor selection when evaluating the effects of xenobiotics on phospholipid synthesis.

Antipeptide antibodies to the 141-1141-1141-1receptor confirm the extracellular orientation of the amino-terminus and the putative first extracellular loop

We developed site-directed rabbit antisera against synthetic peptides selected from the deduced amino acid sequence of the hamster lung beta 2-adrenergic receptor (amino acids 16-31 and 174-189, respectively). All antisera directed against peptide 1 (four of four rabbits) as well as two antisera directed against peptide 2 (two of four rabbits) recognized the purified beta 2-adrenergic receptor in immunoblot conditions when used at a dilution of 1:500. Antisera directed against peptide 1 as well as peptide 2 were able to immunoprecipitate iodinated as well as 125I-cyanopindolol labeled beta 2-adrenergic receptor. This last result implies that the recognized epitopes do not contain the 125I-cyanopindolol binding domain of the beta 2-adrenergic receptor. Immunoblot experiments performed on membrane fractions from hamster lung tissue showed that immunoreactive bands at 64,000, 57,000, 47,000, 44,000 and 38,000 daltons were specifically detected. When purified beta 2-adrenergic receptor was iodinated and submitted to glycolytic and/or tryptic treatments, species with similar molecular weights could be recovered. Then, the immunoreactive bands probably correspond to native beta 2-adrenergic receptor and to degradative or nonglycosylated species of this molecule. The antisera were also able to detect immunoreactive molecules in murine and human cell lines, suggesting conservation of the probed sequences between these species. Enzymatic linked immunosorbent assay tests on intact cells and immunofluorescence studies confirmed that the amino-terminus and putative first extracellular loop are extracellularly located. Immunofluorescence studies on mouse brain primary cultures showed that cells expressing beta 2-adrenergic receptor-like molecules exhibited a neuronal phenotype.

Monoclonal antibodies (G1.4, D7.5) induce secretion of lytic factors from natural killer and T lymphocytes.

Monoclonal antibodies were made by standard procedures using mononuclear cells isolated from hamster lung tissue to immunize the mice. The Moabs (G1.4, D7.5), secreted by the selected hybridomas, were isotyped as IgG2a, Kappa and were lytic in the presence of complement for 20% lymphocytes from lymph nodes or lung tissue; 40-50% of lymphocytes from spleen or bone marrow; and 70-80% of thymocytes or nylon wool nonadherent lung or lymph node lymphocytes. Functional studies showed that prior incubation of either Moab with enriched natural killer (NK) and T lymphocyte subpopulations did not interfere with effector:target conjugate formation but did block the ability of the effector cells to lyse NK-sensitive targets. We conclude that the Moabs bind to a molecule(s) on NK and T lymphocytes. Further, the preincubation medium acquired the capacity to lyse preferentially NK-sensitive targets but not NK-insensitive targets. Interestingly, the Moabs were able to induce secretion of lytic factors from thymocytes and to induce resting peripheral T lymphocytes to acquire NK activity and release lytic factors. The ability of the Moab to signal release of lytic factors was retained even when monovalent Fab fragments were used; therefore, the Moabs initiate secretion by mechanisms that do not apparently involve crosslinking or Fc receptor interaction. G1.4 and D7.5 Moabs were functionally and antigenically crossreactive with human, rat, and hamster lymphocytes but not with mouse lymphocytes. Biochemical and immunochemical analysis showed that the G1.4 and D7.5 antigen on thymus cells and lung mononuclear cells has an apparent molecular weight of 27 kD. Although it is unlikely that there is an universal mechanism for secretion used by cells in general, it is interesting that Moab treatment of three different subpopulation of lymphocytes induced secretion of lytic factors and suggests that under physiologic conditions a common molecule might participate in induction of the secretion pathway. These studies lead to the hypotheses that 1) NK and T lymphocytes share a cell surface molecule that participates in signals that initiate secretion and that 2) the expression of the G1.4, D7.5 antigens precedes thymic differentiation.

In vitro and in vivo modulation of the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in hamster lung tissues

The metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by hamster lung explants was studied. The three major metabolic pathways were α-C-hydroxylation (activation), pyridine N-oxidation (deactivation) and carbonyl reduction. α-C-Hydroxylation and pyridine N-oxidation were linear with time (0.5–5 h) and number of explants per dish (3–10). Addition of [2-(diethylamino)ethyl 2,2-diphenylpentenoate] hydrochloride (SKF-525A) to the culture medium reduced α-C-hydroxylation and pyridine N-oxidation. α-C-Hydroxylation was enhanced by treatment of the hamsters with the two cytochrome P-450 inducers, phenobarbital and 3-methylcholanthrene. These results suggest that cytochrome P-450 monooxygenases are involved in the activation of NNK by α-C-hydroxylation. Three groups of hamsters were fed a control diet or diet supplemented with 2% 2(3)-tert-butyl 4-hydroxyanisole (2(3)-BHA) or given a 0.002% solution of (S)-nicotine to drink for two weeks. Lung explants were then cultured with NNK in vitro. Treatment with 2(3)-BHA and (S)-nicotine induced the α-C-hydroxylation pathways. Pyridine N-oxidation was increased by (S)-nicotine treatment. These results indicate that dietary factors and tobacco smoke components can modulate the metabolism of NNK.