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Recent studies have confirmed that lung microvascular endothelial injury plays a critical role in the pathophysiology of COVID-19. Our group and others have demonstrated the beneficial effects of H2S in several pathological processes and provided a rationale for considering the therapeutic implications of H2S in COVID-19 therapy. Here, we evaluated the effect of the slow-releasing H2S donor, GYY4137, on the barrier function of a lung endothelial cell monolayer in vitro, after challenging the cells with plasma samples from COVID-19 patients or inactivated SARS-CoV-2 virus. We also assessed how the cytokine/chemokine profile of patients’ plasma, endothelial barrier permeability, and disease severity correlated with each other. Alterations in barrier permeability after treatments with patient plasma, inactivated virus, and GYY4137 were monitored and assessed by electrical impedance measurements in real time. We present evidence that GYY4137 treatment reduced endothelial barrier permeability after plasma challenge and completely reversed the endothelial barrier disruption caused by inactivated SARS-CoV-2 virus. We also showed that disease severity correlated with the cytokine/chemokine profile of the plasma but not with barrier permeability changes in our assay. Overall, these data demonstrate that treatment with H2S-releasing compounds has the potential to ameliorate SARS-CoV-2-associated lung endothelial barrier disruption.

Background and purpose During angiogenesis, quiescent endothelial cells (ECs) are activated by various stimuli to form new blood vessels from pre-existing ones in physiological and pathological conditions. Many research groups have shown that hydrogen sulfide (H2 S), the newest member of the gasotransmitter family, acts as a proangiogenic factor. To date, very little is known about the regulatory role of 3-mercaptopyruvate sulfurtransferase (3-MST), an important H2 S-producing enzyme in ECs. The aim of our study was to explore the potential role of 3-MST in human EC bioenergetics, metabolism, and angiogenesis. Experimental approach To assess in vitro angiogenic responses, we used EA.hy926 human vascular ECs subjected to shRNA-mediated 3-MST attenuation and pharmacological inhibition of proliferation, migration, and tube-like network formation. To evaluate bioenergetic parameters, cell respiration, glycolysis, glucose uptake, and mitochondrial/glycolytic ATP production were measured. Finally, global metabolomic profiling was performed to determine the level of 669 metabolic compounds. Key results 3-MST-attenuated ECs subjected to shRNA or pharmacological inhibition of 3-MST significantly reduced EC proliferation, migration, and tube-like network formation. 3-MST silencing also suppressed VEGF-induced EC migration. From bioenergetic and metabolic standpoints, 3-MST attenuation decreased mitochondrial respiration and mitochondrial ATP production, increased glucose uptake, and perturbed the entire EC metabolome. Conclusion and implications 3-MST regulates bioenergetics and morphological angiogenic functions in human ECs. The data presented in the current report support the view that 3-MST pathway may be a potential candidate for therapeutic modulation of angiogenesis. Linked articles This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc.

Human papillomavirus (HPV)-driven oropharyngeal cancer incidence in the United States has steadily increased in the past decades and has now become the most frequently diagnosed HPV-associated cancer type, surpassing cervical cancer. Variations in the HPV genome correlate with tumorigenic risk, and the distribution of genetic variants is extensively studied in cervical cancer, but very little is known about new mutations or the distribution of HPV types and variants in oropharyngeal cancer. Here we present an archival tissue cohort study that compares genomic characteristics of HPV associated with cervical versus oropharyngeal tumors using DNA sequence analysis. We found HPV16 to be more prevalent in oropharyngeal samples than in cervical samples (91.2% versus 52.9%), while HPV18 (1.5% versus 18.2%) and HPV45 (0.7% versus 9.9%) were much less prevalent. Differences between cervix and oropharynx in HPV16 variants distribution were more subtle, but the combined European + Asian (EUR+AS) variant group was more prevalent (90.2% versus 71.4%), while the American Asian 1 + American Asian 2 (AA1+AA2) variant group was much less prevalent (4.4% versus 22.5%) in oropharyngeal cancers. HPV prevalence in oropharyngeal cancers showed an increasing trend from 60% in 2003 to 80% in 2016. We also identified over nine times more nonsynonymous mutations in the HPV E6 gene amplified from oropharyngeal samples, but for E7 the difference in mutation rates between the two anatomical locations was not significant. Overall, we showed that HPV genome in oropharyngeal cancer presents important differences when compared to cervical cancer and this may explain the distinct pathomechanisms and susceptibility to treatment of HPV-associated oropharyngeal cancer.

Background The overall mortality rate in cases of head and neck squamous cell carcinoma (HNSCC) has not improved over the past 30 years, mostly because of the high treatment failure rate among patients with regionally metastatic disease. To better understand the pathobiologic processes leading to lymphatic metastasis development, there is an urgent need for relevant animal models. Methods HNSCC cell lines were implanted into the tongues of athymic nude mice. Histology, immunohistochemistry, and ex vivo 2-photon microscopy were used to evaluate tumor progress and spread. Results Orthotopic xenografts of different HNSCC cell lines produced distinct patterns of survival, tumor histology, disease progression rate, and lymph node metastasis development. Remarkably, all injected cell types reached the lymph nodes within 24 hours after injection, but not all developed metastasis. Conclusion This orthotopic xenograft model closely mimics several characteristics of human cancer and could be extremely valuable for translational studies focusing on lymphatic metastasis development and pathobiology. © 2014 Wiley Periodicals, Inc. Head Neck 36: 1638–1647, 2014

Background The robust desmoplasia associated with head and neck squamous cell carcinoma (HNSCC) suggests that the tumor microenvironment may be an important component in the pathophysiology of this cancer. Moreover, the high recurrence rate and poor clinical response to chemotherapy and radiation treatment further underscores that the non-cancerous cells of the microenvironment, such as mesenchymal stromal cells (MSCs), cancer associated fibroblasts (CAFs), and pericytes, may be important in the pathophysiology of HNSCC. Methods Confocal microscopy and immunohistomchemistry approaches were used to identify MSCs tumor microenvironment from patients with oral cavity and oral pharyngeal squamous cell carcinoma (SCC). In vitro Boyden chamber assays and multiplex magnetic bead assays were used to measure MSC chemotaxis and to identify the chemokines secreted by JHU-011, -012, -019, three cells lines derived from patients with oral pharyngeal SCC. Results We show here that MSCs reside in the tumor microenvironment of patients with oral cavity and oral pharyngeal SCC and are recruited via paracrine mediated tumor cell secretion of (platelet derived growth factor) PDGF-AA. The MSC markers CD90+, CD105+, and gremlin-1+ were found to co-localize on cells within the tumor microenvironment in oral cavity SCC specimens distinct from α-smooth muscle actin staining CAFs. The conditioned media from JHU-011, -012, and -019 caused a significant increase in MSC migration (>60%) and invasion (>50%; p

Age-associated changes in lung structure and function are some of the most important predictors of overall health, cognitive activities and longevity. Common to all aging cells is an increase in oxidatively modified DNA bases, primarily 8-oxo-7,8-dihydroguanine (8-oxoG). It is repaired via DNA base excision repair pathway driven by 8-oxoguanine DNA glycosylase-1 (OGG1-BER), whose role in aging has been the focus of many studies. This study hypothesizes that signaling and consequent gene expression during cellular response to OGG1-BER “wires” senescence/aging processes. To test OGG1-BER was mimicked by repeatedly exposing diploid lung fibroblasts cells and airways of mice to 8-oxoG base. Results showed that repeated exposures led to G1 cell cycle arrest and pre-matured senescence of cultured cells in which over 1000 genes were differentially expressed --86% of them been identical to those in naturally senesced cells. Gene ontology analysis of gene expression displayed biological processes driven by small GTPases, phosphoinositide 3-kinase and mitogen activated kinase cascades both in cultured cells and lungs. These results together, points to a new paradigm about the role of DNA damage and repair by OGG1 in aging and age-associated disease processes.

The pathomechanism of Alzheimer's disease (AD) is multifactorial although the most popular hypotheses are centered on the effects of the misfolded, aggregated protein, amyloid beta (Abeta) and on Tau hyperphosphorylation.Double blinded clinical trials were planned to demonstrate the effect of Colostrinin (CLN) on instrumental daily activities of AD patients. The potential molecular mechanisms by which CLN mediates its effects were investigated by gene expression profiling.RNAs isolated from CLN-treated cells were analyzed by high-density oligonucleotide arrays. Network and pathway analyses were performed using the Ingenuity Pathway Analysis software.The Full Sample Analysis at week 15 showed a stabilizing effect of CLN on cognitive function in ADAS-cog (p = 0.02) and on daily function in IADL (p = 0.02). The overall patient response was also in favor of CLN (p = 0.03). Patients graded as mild on entry also showed a superior response of ADAS-cog compared to more advanced cases (p = 0.01). Data derived from microarray network analysis show that CLN elicits highly complex and multiphasic changes in the cells' transcriptome. Importantly, transcriptomal analysis showed that CLN alters gene expression of molecular networks implicated in Abeta precursor protein synthesis, Tau phosphorylation and increased levels of enzymes that proteolitically eliminate Abeta. In addition, CLN enhanced the defense against oxidative stress and decreased expression of inflammatory chemokines and cytokines, thereby attenuating inflammatory processes that precede Alzheimer's and other neurological diseases.Together these data suggest that CLN has promising potential for clinical use in prevention and therapy of Alzheimer's and other age-associated central nervous system diseases.

Colostrinin (CLN) is a uniform mixture of low-molecular weight proline-rich polypeptides isolated from the mother's first milk, colostrum. Exposure of cells to CLN decreases intracellular levels of reactive oxygen species by regulating glutathione metabolism and modulating activities of antioxidant enzymes and mitochondrial function. It also inhibits beta amyloid-induced apoptosis and induces neurite outgrowth of pheochromocytoma cells. Administration of CLN to Alzheimer's disease patients has resulted in a stabilizing effect on cognitive function. We analyzed CLN-induced gene expression changes using high-density oligonucleotide arrays and transcriptomal network analysis. We found that CLN elicited highly complex and multiphasic changes in the gene expression profile of treated cells. CLN treatment affected a total of 58 molecular networks, 27 of which contained at least 10 differentially expressed genes. Here we present CLN-modulated gene networks as potential underlying molecular mechanisms leading to the reported effects of CLN on cellular oxidative state, chemokine and cytokine production, and cell differentiation, as well as on pathological processes like allergy, asthma, Alzheimer's, and other neurological diseases. Based on our results, we also predict possible modulatory effects of CLN on adipocytokine gene networks that play a crucial role in the pathobiology of diabetes, cardiovascular disorders, obesity, and inflammation. Taken together, CLN-altered gene expression networks presented here provide the molecular basis for previously described biological phenomena and predict potential fields of application for CLN in the prevention and treatment of diseases.

Summary Objectives : The hepatocyte growth factor receptor (Met) is frequently overexpressed in Head and Neck Squamous Cell Carcinoma (HNSCC), correlating positively with high-grade tumors and shortened patient survival. As such, Met may represent an important therapeutic target. The purpose of this study was to explore the role of Met signaling for HNSCC growth and locoregional dissemination. Materials and methods : Using a lentiviral system for RNA interference, we knocked down Met in established HNSCC cell lines that express high levels of the endogenous receptor. The effect of Met silencing on in vitro proliferation, cell survival and migration was examined using western analysis, immunohistochemistry and live cell imaging. In vivo tumor growth, dissemination and mouse survival was assessed using an orthotopic tongue mouse model for HNSCC. Results : We show that Met knockdown (1) impaired activation of downstream MAPK signaling; (2) reduced cell viability and anchorage independent growth; (3) abrogated HGF-induced cell motility on laminin; (4) reduced in vivo tumor growth by increased cell apoptosis; (5) caused reduced incidence of tumor dissemination to regional lymph nodes and (6) increased the survival of nude mice with orthotopic xenografts. Conclusion : Met signaling is important for HNSCC growth and locoregional dissemination in vivo and that targeting Met may be an important strategy for therapy.

Accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) in the DNA results in genetic instability and mutagenesis, and is believed to contribute to carcinogenesis, aging processes and various aging-related diseases. 8-OxoG is removed from the DNA via DNA base excision repair (BER), initiated by 8-oxoguanine DNA glycosylase-1 (OGG1). Our recent studies have shown that OGG1 binds its repair product 8-oxoG base with high affinity at a site independent from its DNA lesion-recognizing catalytic site and the OGG1•8-oxoG complex physically interacts with canonical Ras family members. Furthermore, exogenously added 8-oxoG base enters the cells and activates Ras GTPases; however, a link has not yet been established between cell signaling and DNA BER, which is the endogenous source of the 8-oxoG base. In this study, we utilized KG-1 cells expressing a temperature-sensitive mutant OGG1, siRNA ablation of gene expression, and a variety of molecular biological assays to define a link between OGG1-BER and cellular signaling. The results show that due to activation of OGG1-BER, 8-oxoG base is released from the genome in sufficient quantities for activation of Ras GTPase and resulting in phosphorylation of the downstream Ras targets Raf1, MEK1,2 and ERK1,2. These results demonstrate a previously unrecognized mechanism for cellular responses to OGG1-initiated DNA BER.

Extracellular matrix factors within the tumor microenvironment that control resistance to chemotherapeutics are poorly understood. This study focused on understanding matrix adhesion pathways that control the oral carcinoma response to cisplatin. Our studies revealed that adhesion of HN12 and JHU012 oral carcinomas to carcinoma matrix supported tumor cell proliferation in response to treatment with cisplatin. Proliferation in response to 30 µM cisplatin was not observed in HN12 cells adherent to other purified extracellular matrices such as Matrigel, collagen I, fibronectin or laminin I. Integrin β1 was important for adhesion to carcinoma matrix to trigger proliferation after treatment with cisplatin. Disruption of talin expression in HN12 cells adherent to carcinoma matrix increased cisplatin induced proliferation. Pharmacological inhibitors were used to determine signaling events required for talin deficiency to regulate cisplatin induced proliferation. Pharmacological inhibition of NF-kB reduced proliferation of talin-deficient HN12 cells treated with 30 µM cisplatin. Nuclear NF-kB activity was assayed in HN12 cells using a luciferase reporter of NF-kB transcriptional activity. Nuclear NF-kB activity was similar in HN12 cells adherent to carcinoma matrix and collagen I when treated with vehicle DMSO. Following treatment with 30 µM cisplatin, NF-kB activity is maintained in cells adherent to carcinoma matrix whereas NF-kB activity is reduced in collagen I adherent cells. Expression of talin was sufficient to trigger proliferation of HN12 cells adherent to collagen I following treatment with 1 and 30 µM cisplatin. Talin overexpression was sufficient to trigger NF-kB activity following treatment with cisplatin in carcinoma matrix adherent HN12 cells in a process disrupted by FAK siRNA. Thus, adhesions within the carcinoma matrix create a matrix environment in which exposure to cisplatin induces proliferation through the function of integrin β1, talin and FAK pathways that regulate NF-kB nuclear activity.

Most flow/image cytometric data analysis methods look for clusters in the data corresponding to specific cell subpopulations. Comparisons between different cytometry datafiles often use human pattern recognition visualization of all the different combinations of variables ("parameters") two at a time in so-called bivariate scattergrams. Not only is this tedious, but it can miss potential clusters due to projection of higher dimensional dataspaces down onto two dimensional planes making them indiscernible as separate clusters. Novel data mining algorithms, implemented in software allow for the comparison of two or more higher dimensional datafiles without the requirement for reduction of dimensionality for human visualization. Of equal importance is the comparison of higher dimensional clusters which may move around slightly in space, yet still be "similar" according to algorithms which provide measures of similarity. This software, written in C/C++ and currently implemented in software with a Windows graphical user interface, allows for direct reading of FCS2.0 format flow cytometry datafiles of any number of parameters. In a few minutes or less, complex multiparameter data of two or more files can be compared on a personal computer or workstation. The software operates in either supervised or unsupervised mode, depending on whether the user wishes to include prior user knowledge or in a data mining discovery mode. Differences between these files can be exported as sub-datafiles which can be further analyzed using any other software that can read FCS2.0 data format.

Background: Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAP™ (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection). Methods: LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy. Results: Live cell samples (adherent and suspension) could be purified to 90–100% purity with 50–90% yield, causing minimal cell damage depending on the cell type and plating density. Nearly one hundred percent of the targeted cells of all cell types examined could be successfully optoinjected with dextrans of 3–70 kDa, causing no visual damage to the cells. Indirect optoinjection effects were observed on untargeted cells within 5–60 μm to targeted areas under conditions used here. Conclusions: LEAP provides solutions in cell purification and targeted macromolecule delivery for traditional and challenging applications where other methods fall short. © 2006 International Society for Analytical Cytology

A novel data mining program called “subtractive clustering” picks out the most important differences between two or more flow cytometry listmode data files. While making no assumptions about the data, the program uses a variable weight and skew metric in the determination of bin size allowing for subtractive clustering of data without the need for bit-reduction or projection. In contrast, other subtraction methods, such as channel-by-channel subtraction, are dependent upon dimensionality and resolution, which can lead to an overestimation of positive cells because they do not account for the overall distribution of the test and control data sets. By taking into account human visual inspection of the data it is possible for the experimenter to choose an optimal subtraction by choosing an appropriate weight and skew metric, but without allowing direct modification of the results. By maximizing a bin size which can still differentiate clusters, it is possible to minimize computation while still removing data. The choice of control weight allows for different levels of bin destruction during the subtraction stage, the smaller the number the more conservative the subtraction, the larger, the more liberal. Three data sets illustrate full dimensional subtraction, single step biological data and multi-stage subtraction to show definitive test results. Subtractive clustering was able to conservatively remove control information leaving populations of interest. Subtractive clustering provides a powerful comparison of clusters and is a first step for finding non-obvious (hidden) differences and minimizing human prejudice during the analysis.

Background: Most biological samples are cell mixtures. Some basic questions are still unanswered about analyzing these heterogeneous samples using gene expression microarray technology (MAT). How meaningful is a cell mixture’s overall gene expression profile (GEP)? Is it necessary to purify the cells of interest before microarray analysis, and how much purity is needed? How much does the purification itself distort the GEP, and how well can the GEP of a small cell subset be recovered? Methods: Model cell mixtures with different cell ratios were analyzed by both spotted and Affymetrix MAT. GEP distortion during cell purification and GEPs of purified cells were studied. CD34 cord blood cells were purified and analyzed by MAT. Results: GEPs for mixed cell populations were found to mirror the cell ratios in the mixture. Over 75% pure samples were indistinguishable from pure cells by their overall GEP. Cell purification preserved the GEP. The GEPs of small cell subsets could be accurately recovered by cell sorting both from model cell mixtures and from cord blood. Conclusions: Purification of small cell subsets from a mixture prior to MAT is necessary for meaningful results. Even completely hidden GEPs of small cell subpopulations can be recovered by cell sorting. © 2004 Wiley-Liss, Inc.

Gene expression microarray analyses of mixtures of cells approximate a weighted average of the gene expression profiles (GEPs) of each cell type according to its relative abundance in the overall cell sample being analyzed. If the targeted subpopulation of cells is in the minority, or the expected perturbations are marginal, then such changes will be masked by the GEP of the normal/unaffected cells. We show that the GEP of a minor cell subpopulation is often lost when that cell subpopulation is of a frequency less than 30 percent. The GEP is almost always masked by the other cell subpopulations when that frequency drops to 10 percent or less. On the basis of these results one should always assume that the GEP of a given cell subpopulation is probably seriously affected by, the presence of significant numbers of other "contaminating" cell types. Several methodologies can be employed to enrich the target cells submitted for microarray analyses. These include magnetic sorting and laser capture microdissection. If a cell subpopulation of interest is small, very high-throughput cell separation technologies are needed to separate enough cells for conventional microarrays. However, high-throughput flow cytometry/cell sorting overcomes many restrictions of experimental enrichment conditions. This technology can also be used to sort smaller numbers of cells of specific cell subpopulations and subsequently amplify their mRNAs before microarray analyses. When purification techniques are applied to unfixed samples, the potential for changes in gene levels during the process of collection is an additional concern. Since RNA rapidly degrades, and specific mRNAs turn over in minutes or hours, the cell separation process must be very rapid. Hence, high-throughput cell separation (HTS) technologies are needed that can process the necessary number of cells expeditiously in order to avoid such uncontrolled changes in the target cells GEP. In cases where even the use of HTS yields only a small number of cells, the mRNAs (after reverse transcription to cDNA's) must be amplified to yield enough material for conventional microarray analyses. However, the problem of using "microamplification" PCR methods to expand the amount of cDNAs (from mRNAs) is that it is very difficult to amplify equally all of the mRNAs. Unequal amplification leads to a distorted gene expression profile on the microarray. Linear amplifications is difficult to achieve. Unfortunately, present-day gene-chips need to be about 100 times more sensitive than they are now to be able to do many biologically and biomedically meaningful experiments and clinical tests.

A number of theoretical and practical limits of high-speed flow cytometry/cell sorting are important for clinical diagnostics and therapeutics. Three applications include: (1) stem cell isolation with tumor purging for minimal residual disease monitoring and treatment, (2) identification and isolation of human fetal cells from maternal blood for prenatal diagnostics and in-vitro therapeutics, and (3) high-speed library screening for recombinant vaccine production against unknown pathogens.

The natural hormone estradiol (E2) induces tumors in rodents and various types of DNA damage in vitro and in vivo, but has not been mutagenic in bacterial or mammalian assays. Recent reports of chromosomal and genetic lesions induced by E2 has led us to re-examine the mutation frequency and molecular alterations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in Chinese hamster V79 cells. E2 at both physiological and pharmacological concentrations (10-11, 10-10, and 10-7, 10-6 M) significantly increased the mutation frequency of the hprt gene by 2. 57-, 3.45-, 2.63-, and 8.78-fold, respectively, compared to the controls, while 10-13, 10-12, 10-9, or 10-8 M E2 induced little change (< or =0.93-fold). PCR and a molecular analysis of the hprt coding sequence identified genetic lesions in the cDNA and/or genomic DNA in 15 of the 21 picked E2-induced mutants (71%). Simple base substitutions, such as Tright curved arrow G or Tright curved arrow A transversions, were the most common mutations (8/21 or 38%) and frequently occurred at 122 bp or 407 bp of the hprt coding sequence. Deletion mutations were detected in 6 of the 21 clones (29%). An Aright curved arrow G and a Cright curved arrow T transition and a four-base insertion (TATT) were identified each in one mutant clone. A RT-PCR analysis demonstrated an abundant expression of the estrogen receptor-alpha (ERalpha). However, ICI 182,780, an antagonist of ERalpha, acted in an additive manner with E2 and increased the hprt mutation frequency. In conclusion, E2 induces a low frequency of mutations (deletions and point mutations) in V79 cells, which is consistent with the weak carcinogenic activity of this hormone. The mutagenic effects of E2 in V79 cells are not mediated by the ERalpha.

This is the first study documenting the induction of gamma-interferon (IFN-gamma) in human embryonic fibroblasts during human cytomegalovirus (HCMV) replication. Infection of cells with HCMV resulted in the consistent production of IFN-gamma RNA, as determined by RT-PCR and Northern blot analysis. Western blot analysis of cell lysates and immunoprecipitates from the cultural fluids of infected cells demonstrated the presence of IFN-gamma at the protein level. Induction of IFN-gamma required infectious HCMV, since high-dose ultraviolet inactivation of the virus stock eliminated IFN-gamma production. Further, IFN-gamma induction appears to be a late event in the virus replication cycle, since inhibition of HCMV DNA synthesis (e.g., phosphonoacetic acid) blocked the increase in IFN-gamma. Soluble factor(s) released from HCMV-infected cells apparently did not contribute to the induction of IFN-gamma, since virus stocks from which virus had been removed by sedimentation did not induce production of IFN-gamma. The appearance of IFN-gamma at late stages of HCMV infection and its elimination in the presence of an inhibitor (Actinomycin D) of RNA synthesis indicate a true transcriptional induction of this lymphokine at the RNA and protein levels. The significance of IFN-gamma production with regard to the replication and pathogenesis of HCMV in vitro and in vivo will require further investigation.