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Introduction: The COVID-19 pandemic has become a serious challenge for humanity almost everywhere globally. Despite active vaccination around the world, the incidence proportion in different countries varies significantly as of May 2022. The reason may be a combination of demographic, immunological, and epidemiological factors. The purpose of this study was to analyze possible relationships between COVID-19 incidence proportion in the population and the types of SARS-CoV-2 vaccines used in different countries globally, taking into account demographic and epidemiological factors. Materials and methods: An initial database was created of demographic and immunoepidemiological information about the COVID-19 situation in 104 countries collected from published official sources and repository data. The baseline included, for each country, population size and density; SARS-CoV-2 testing coverage; vaccination coverage; incidence proportion; and a list of vaccines that were used, including their relative share among all vaccinations. Subsequently, the initial data set was stratified by population and vaccination coverage. The final data set was subjected to statistical processing both in general and taking into account population testing coverage. Results: After formation of the final data set (including 53 countries), it turned out that reported COVID-19 case numbers correlated most strongly with testing coverage and the proportions of vaccine types used, specifically, mRNA (V1); vector (V2); peptide/protein (V3); and whole-virion/inactivated (V4). Due to the fact that an inverse correlation was found between 'reported COVID-19 case numbers' with V2, V3, and V4, these three vaccine types were also combined into one analytic group, 'non-mRNA group' vaccines (Vnmg). When the relationship between vaccine type and incidence proportion was examined, minimum incidence proportion was noted at V1:Vnmg ratios (%:%) from 0:100 to 30:70. Maximum incidence proportion was seen with V1:Vnmg from 80:20 to 100:0. On the other hand, we have shown that the number of reported COVID-19 cases in different countries largely depends on testing coverage. To offset this factor, countries with low and extremely high levels of testing were excluded from the data set; it was then confirmed that the largest number of reported COVID-19 cases occurred in countries with a dominance of V1 vaccines. The fewest reported cases were seen in countries with a dominance of Vnmg vaccines. Conclusion: In this paper, we have shown for the first time that the level of reported COVID-19 incidence proportion depends not only on SARS-CoV-2 testing and vaccination coverage, which is quite logical, but probably also on the vaccine types used. With the same vaccination level and testing coverage, those countries that predominantly use vector and whole-virion vaccines feature incidence proportion that is significantly lower than countries that predominantly use mRNA vaccines.

The work is devoted to the study of the structural characteristics of the myeloperoxidase-ceruloplasmin-thrombin complex using small-angle neutron scattering methods in combination with computer modeling, as well as surface plasmon resonance and solid-phase enzyme assay. We have previously shown that the functioning of active myeloperoxidase during inflammation, despite the presence in the blood of an excess of ceruloplasmin which inhibits its activity, is possible due to the partial proteolysis of ceruloplasmin by thrombin. In this study, the myeloperoxidase-ceruloplasmin-thrombin heterohexamer was obtained in vitro. The building of a heterohexamer full-atomic model in silico, considering the glycosylation of the constituent proteins, confirmed the absence of steric barriers for the formation of protein-protein contacts. It was shown that the partial proteolysis of ceruloplasmin does not affect its ability to bind to myeloperoxidase, and a structural model of the heterohexamer was obtained using the small-angle neutron scattering method., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction: Respiratory infections, collectively, are one of the World's most common and serious illness groups. As recent observations have shown, the most severe courses of acute respiratory infection, often leading to death, are due to uncontrolled cytokine production (hypercytokinemia)., Methods: The study involved 364 patients with respiratory illness being treated in clinics in St. Petersburg (Russia) in 2018-2019 and 30 healthy controls. Cytokine analysis was carried out in the acute phase of illness (2-3 days from onset of initial symptoms) and in the stage of recovery (days 9-10). The research presented is devoted to the assessment of mRNA expression of specific cytokines (interleukin [IL]-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, tumor necrosis factor-α [TNF-α], and interferon-λ) and MxA in whole blood leukocytes, by means of real-time polymerase chain reaction., Results: In 70% of patients, bacterial or viral pathogens were identified, with influenza viral infections (types A and B) prevailing. Significant increases in the expression of IL-18, TNF, and IL-10 were observed, relative to controls, only with influenza viral infections. We have shown a difference in IL-6 mRNA expression in patients with bacterial or viral pathogens. No statistically significant difference was found in white blood cells IL-4 expression levels between patients and healthy controls., Conclusion: Investigation of the nuances of systemic cytokine production, in response to specific viral and bacterial pathogens, makes it possible to assess the risks of developing hypercytokinemia during respiratory infection with agents circulating in the human population and to predict the pathogenicity and virulence of circulating threats., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Global Infectious Diseases.)

The aggressive infectious nature of SARS-CoV-2, its rapid spread, and the emergence of mutations necessitate investigation of factors contributing to differences in SARS-CoV-2 susceptibility and severity. The role of genetic variations in the human HLA continues to be studied in various populations in terms of both its effect on morbidity and clinical manifestation of illness. The study included 484 COVID-19 convalescents (northwest Russia residents of St. Petersburg). Cases in which the responsible strain was determined were divided in two subgroups: group 1 ( n = 231) had illness caused by genovariants unrelated to variant of concern (VOC) strains; and group 2 ( n = 80) had illness caused by the delta (B.1.617.2) VOC; and a control group ( n = 1456). DNA typing (HLA-A, B, DRB1) was performed at the basic resolution level. HLA-A*02 was associated with protection against infection caused by non-VOC SARS-CoV-2 genetic variants only but not against infection caused by delta strains. HLA-A*03 was associated with protection against infection caused by delta strains; and allele groups associated with infection by delta strains were HLA-A*30, B*49, and B*57. Thus, in northwest Russia, HLA-A*02 was associated with protection against infection caused by non-VOC SARS-CoV-2 genetic variants but not against delta viral strains. HLA-A*03 was associated with a reduced risk of infection by delta SARS-CoV-2 strains. HLA-A*30, HLA-B*49, and HLA-B*57 allele groups were predisposing factors for infection by delta (B.1.617.2) strains.

In this review, we analyze the epidemiological and ecological features of influenza B, one of the most common and severe respiratory infections. The review presents various strategies for cross-protective influenza B vaccine development, including recombinant viruses, virus-like particles, and recombinant proteins. We provide an overview of viral proteins as cross-protective vaccine targets, along with other updated broadly protective vaccine strategies. The importance of developing such vaccines lies not only in influenza B prevention, but also in the very attractive prospect of eradicating the influenza B virus in the human population.

In this work, we have extended our previously proposed approach for determining protein concentrations in human serum (using MALDI-TOF mass spectrometry) to include simultaneous analysis of several proteins associated with acute inflammation (alpha-2-macroglobulin, fetuin-A, serum amyloid A1). This technique can be used to diagnose systemic inflammation and provides results in 4-5 h. The developed approach was verified using standard immunological methods (ELISA). Samples from 87 individuals, in specific groups, were used for testing and validation: control; inflammatory soft tissue disease accompanied by sepsis; influenza A infection; or COVID-19. The feasibility of differentiating patient groups with the aforementioned conditions was analyzed using a combination of the inflammatory markers described. For fetuin-A and serum amyloid A1, diagnostically significant concentration ranges were established., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

An essential part of designing any biotechnological process is examination of the physiological state of producer cells in different phases of cultivation. The main marker of a bacterial cell's state is its fatty acid (FA) profile, reflecting membrane lipid composition. Consideration of FA composition enables assessment of bacterial responses to cultivation conditions and helps biotechnologists understand the most significant factors impacting cellular metabolism. In this work, soil SDS-degrading Pseudomonas helmanticensis was studied at the fatty acid profile level, including analysis of rearrangement between planktonic and aggregated forms. The set of substrates included fat hydrolysates, SDS, and their mixtures with glucose. Such media are useful in bioplastic production since they can help incrementally lower overall costs. Conventional gas chromatography-mass spectrometry was used for FA analysis. Acridine orange-stained aggregates were observed by epifluorescence microscopy. The bacterium was shown to change fatty acid composition in the presence of hydrolyzed fats or SDS. These changes seem to be driven by the depletion of metabolizable substrates in the culture medium. Cell aggregation has also been found to be a defense strategy, particularly with anionic surfactant (SDS) exposure. It was shown that simple fluidity indices (such as saturated/unsaturated FA ratios) do not always sufficiently characterize a cell's physiological state, and morphological examination is essential in cases where complex carbon sources are used., (© 2021. The Microbiological Society of Korea.)

The influenza NS1 protein is involved in suppression of the host immune response. Recently, there is growing evidence that prion-like protein aggregation plays an important role in cellular signaling and immune responses. In this work, we obtained a recombinant, influenza A NS1 protein and showed that it is able to form amyloid-like fibrils in vitro. Using proteolysis and subsequent mass spectrometry, we showed that regions resistant to protease hydrolysis highly differ between the native NS1 form (NS1-N) and fibrillar form (NS1-F); this indicates that significant structural changes occur during fibril formation. We also found a protein fragment that is capable of inducing the process of fibrillogenesis at 37 °C. The discovery of the ability of NS1 to form amyloid-like fibrils may be relevant to uncovering relationships between influenza A infection and modulation of the immune response., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Two influenza A nucleoprotein variants (wild-type: G102R; and mutant: G102R and E292G) were studied with regard to macro-molecular interactions in oligomeric form (24-mers). The E292G mutation has been previously shown to provide cold adaptation. Molecular dynamics simulations of these complexes and trajectory analysis showed that the most significant difference between the obtained models was distance between nucleoprotein complex strands. The isolated complexes of two ribonucleoprotein variants were characterized by transmission electron microscopy and differential scanning fluorimetry (DSF). Presence of the E292G substitution was shown by DSF to affect nucleoprotein complex melting temperature. In the filament interface peptide model, it was shown that the peptide corresponding in primary structure to the wild-type NP (SGYDF E REGYS) is prone to temperature-dependent self-association, unlike the peptide corresponding to E292G substitution (SGYDF G REGYS). It was also shown that the SGYDF E REGYS peptide is capable of interacting with a monomeric nucleoprotein (wild type); this interaction's equilibrium dissociation constant is five orders of magnitude lower than for the SGYDF G REGYS peptide. Using small-angle neutron scattering (SANS), the supramolecular structures of isolated complexes of these proteins were studied at temperatures of 15, 32, and 37 °C. SANS data show that the structures of the studied complexes at elevated temperature differ from the rod-like particle model and react differently to temperature changes. The data suggest that the mechanism behind cold adaptation with E292G is associated with a weakening of the interaction between strands of the ribonucleoprotein complex and, as a result, the appearance of inter-chain interface flexibility necessary for complex function at low temperature.Communicated by Ramaswamy H. Sarma.

Introduction: Influenza A virus infection can lead to endothelial dysfunction (ED), including apoptosis of endothelial cells and modulation of endothelial factor activities. Affected biochemical factors may include those playing important roles in vascular homeostasis. However, the effect of this pathogen on the expression pattern of key endothelial factors is still unknown.The aim of this work was to study the expression of endothelial nitric oxide synthase (eNOS) and plasminogen activator inhibitor-1 (PAI-1, serpin E1) in the EA.hy926 endothelial cells., Research Objectives: to assess expression of eNOS and PAI-1 in endothelial cells infected with influenza virus A(H1N1)pdm09, and to identify homologous fragments in structure of viral proteins and endothelial factors., Material and Methods: Cells were infected with influenza virus A/St. Petersburg/48/16 (H1N1)pdm09 and analyzed in dynamics in 6, 12, 18, 24, 48, and 72 hrs post infection (hpi). Detection of endothelial factors expression levels was performed by immunocytochemical method (ICC) using antibodies for eNOS and PAI-1 while quantitative assessment of expression levels was carried out by program Nis-Elements F3.2 («Nikon», Japan). The search for homologous sequences between viral proteins and eNOS and PAI-1 was performed by computer comparison. Sequences were analyzed as fragments 12 amino acid residues (aar) in length., Results and Discussion: eNOS expression in infected cells had decreased to 7.9% by 6 hpi (control was taken as 100%) to 3.3% at 72 hpi. PAI-1 expression varied significantly over the course of the experiment: by 6 hpi it had decreased to 49.6%, and to 43.2% by 12 hpi. Later PAI-1 levels were: 116.3% (18 hpi); 18.9% (24 hpi); 23.5% (48 hpi), and 35% (72 hpi)., Conclusion: These results indicate that influenza A infection of endothelial cells causes a significant decrease in eNOS expression, while modulating PAI-1 one. The described phenomenon can be used in the further development of directions of pathogenetic therapy of vascular complications of infection caused by this pathogen.

One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M's 'bait region', and the peptide 705 VGFYESDVMGR 715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue ( 18 O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin's modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions., Competing Interests: Declaration of competing interest We have no conflict of interest to declare., (Copyright © 2021 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Interferons (IFN) are crucial for the innate immune response. Slightly more than two decades ago, a new type of IFN was discovered: the lambda IFN (type III IFN). Like other IFN, the type III IFN display antiviral activity against a wide variety of infections, they induce expression of antiviral, interferon-stimulated genes ( MX1 , OAS , IFITM1 ), and they have immuno-modulatory activities that shape adaptive immune responses. Unlike other IFN, the type III IFN signal through distinct receptors is limited to a few cell types, primarily mucosal epithelial cells. As a consequence of their greater and more durable production in nasal and respiratory tissues, they can determine the outcome of respiratory infections. This review is focused on the role of IFN-λ in the pathogenesis of respiratory viral infections, with influenza as a prime example. The influenza virus is a major public health problem, causing up to half a million lethal infections annually. Moreover, the virus has been the cause of four pandemics over the last century. Although IFN-λ are increasingly being tested in antiviral therapy, they can have a negative influence on epithelial tissue recovery and increase the risk of secondary bacterial infections. Therefore, IFN-λ expression deserves increased scrutiny as a key factor in the host immune response to infection.

Upper respiratory tract infections are the world's most common infectious disease. The etiologic agents behind upper respiratory tract infections (URTIs) are, in fact, a diverse set of pathogens such as influenza, parainfluenza, adenovirus, rhinovirus, and others. More than 200 pathogens are known to be involved. Differential diagnosis of viral infections is sometimes complicated by their diversity or similarity of clinical presentation. This work is devoted to the development of a method which enables simultaneous detection of six common viral URTI pathogens: IAV; IBV; RSV; hAdV; hPIV2; and hPIV3. Antibody microarray technology is utilized to accomplish the analysis. In preparation for protein microchip creation, we produced, characterized, and selected approximately 50 monoclonal antibodies; for each of the aforementioned pathogens, an optimal monoclonal antibody pair was selected. A protein microchip was created, and its core working conditions were optimized. With a balance between convenience and maximal assay sensitivity in mind, a one-step analysis approach was developed for accomplishing the ELISA-like "sandwich" interaction on the manufactured microchip (antibody microarray). Reference viral strains were used to establish the lower limits of detection (LoD) for the assay. For IAV, the LoD was 0.25 ng/ml total viral protein. For other viruses, the LoD ranged from 1 to 2 ng/ml total protein. These sensitivity limits are slightly better than those of standard ELISA, but inferior to those of PCR. Overall, we believe that the developed microchip is a good alternative to existing methods, allowing relatively quick (overnight), inexpensive, simultaneous screening of several pathogens. The design of the antibody microarray is conducive to further development, and the panel of analyzed pathogens can be expanded to include approximately 50 members., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Mechanistic target of rapamycin (MTOR) is a highly conserved serine/threonine kinase that critically regulates cell growth, proliferation, differentiation, and survival. Previously, we have implicated Mtor as a plasmacytoma-resistance locus, Pctr2 , in mice. Here, we report that administration of the tumor-inducing agent pristane decreases Mtor gene expression to a greater extent in mesenteric lymph nodes of BALB/cAnPt mice than of DBA/2N mice. We identified six allelic variants in the Mtor promoter region in BALB/cAnPt and DBA/2N mice. To determine the effects of these variants on Mtor transcription, we constructed a series of luciferase reporters containing these promoter variants and transfected them into mouse plasmacytoma cells. We could attribute the differences in Mtor promoter activity between the two mouse strains to a C → T change at the -6 position relative to the transcriptional start site Tssr 40273; a T at this position in the BALB promoter creates a consensus binding site for the transcription factor MZF1 (myeloid zinc finger 1). Results from electrophoretic mobility shift assays and DNA pulldown assays with ChIP-PCR confirmed that MZF1 binds to the cis -element TGGGGA located in the -6/-1 Mtor promoter region. Of note, MZF1 significantly and differentially down-regulated Mtor promoter activity, with MZF1 overexpression reducing Mtor expression more strongly in BALB mice than in DBA mice. Moreover, MZF1 overexpression reduced Mtor expression in both fibroblasts and mouse plasmacytoma cells, and Mzf1 knockdown increased Mtor expression in BALB3T3 and NIH3T3 fibroblast cells. Our results provide evidence that MZF1 down-regulates Mtor expression in pristane-induced plasmacytomas in mice.

The erythrocyte sedimentation rate (ESR) has become a ubiquitously used technique in medicine as a marker of systemic illness. The test involves placing anticoagulated whole blood into an upright test tube and monitoring the rate at which red blood cells (RBC) fall over time. Negative charges keep RBC from sticking together. If this charge is neutralised, RBC stack into chains, or rouleaux, and fall more rapidly. ESR can be measured with a variety of tests: Westergren and modified Westergren; Wintrobe; micro-ESR. The Westergren is the most commonly used method of performing the ESR. Technical factors, such as temperature, time from specimen collection, tube orientation and vibration, can affect the results. RBC size, shape and concentration impact the ESR. Plasma characteristics are also important determinants of the ESR. Other factors that can change ESR include age, sex, race, medications and disease states, such as obesity, hypofibrinogenaemia and congestive heart failure. Other acute-phase reactants besides the ESR include C-reactive protein, fibrinogen, complement, ferritin, plasma viscosity, serum amyloid A and albumin. When clinical suspicion for infection or inflammation is low, a normal ESR can reassure that there is no active disease. The slow rise (48 h) and fall of the ESR relative to other acute-phase reactants may make it superior for monitoring inflammation in more chronic conditions. In conjunction with physical findings and other laboratory values, the ESR value can be used to screen for disease or disease complications, aid in disease diagnosis or assess disease activity or response to therapy., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Oral chronic graft-versus-host disease (cGVHD) is a frequent, clinically significant sequela of allogeneic hematopoietic stem cell transplantation (HSCT). This study was designed to elucidate relationships among clinical characteristics of oral cGVHD and related oral pain and oral dryness, salivary proinflammatory cytokine interleukin (IL)-6 and IL-1alpha concentrations, and health-related quality of life (HRQL). An understanding of the characteristics and correlates of oral cGVHD manifestations and related symptoms, such as oral dryness, is fundamental to the development of therapeutic interventions. Oral cGVHD severity was assessed with the Oral Mucositis Rating Scale (OMRS). Oral pain and perceived intensity of oral dryness were self-reported via a visual analog scale and a numeric rating scale, respectively. HRQL was assessed with the Functional Assessment of Cancer Therapy-General (FACT-G). Salivary IL-1alpha and IL-6 concentrations were measured by enzyme-linked immunosorbent assay. All 42 adult subjects (59% males) had clinician-assessed oral cGVHD by the OMRS scale (mean score, 18.38 +/- 12.99; range, 2-46). Oral dryness (in 43% of subjects; mean OMRS score, 2.56 +/- 3.45; range, 0-10) was more prevalent than oral pain (8%; mean score, 0.13 +/- 0.47). Salivary IL-6 was associated with oral cGVHD severity (r = 0.49; P < .01), oral ulceration (r = 0.38; P = .04), and erythema (r = 0.63; P < .01). FACT-G total score and physical and emotional well-being subscale scores were meaningfully lower than U.S. population normative values. Participants with more severe oral cGVHD manifestations had significantly inferior social/family well being (r = -0.49; P < .01). Oral dryness was associated with higher salivary IL-1alpha (r = 0.41; P = .04) and, controlling for cGVHD severity, with lower HRQL (r = -0.41; P = .03). Subjects with moderate to severe oral dryness tended to report the poorest overall HRQL. This study provides preliminary evidence of the relationship between oral dryness and HRQL, the contribution of oral cGVHD to inferior HRQL, and the association between IL-6 and oral cGVHD severity, ulceration, and erythema. The high prevalence of oral dryness and its relationship to HRQL in a sample of subjects with oral cGVHD underscores the importance of improving our evaluation and management of this symptom in long-term survivors of allogeneic HSCT. The positive associations between IL-6 and oral cGVHD severity and erythema, as well as the positive trend with oral ulceration, warrant further exploration of this cytokine as a potential biomarker of active oral cGVHD.

New insights into the biological properties of cyclooxygenase-2 (COX-2) and its response pathway challenge the hypothesis that COX-2 is simply pro-inflammatory and inhibition of COX-2 solely prevents the development of inflammation and ameliorates inflammatory pain. The present study performed a comprehensive analysis of gene/protein expression induced by a selective inhibitor of COX-2, rofecoxib, compared with a non-selective COX inhibitor, ibuprofen, and placebo in a clinical model of acute inflammatory pain (the surgical extraction of impacted third molars) using microarray analysis followed by quantitative RT-PCR verification and Western blotting. Inhibition of COX-2 modulated gene expression related to inflammation and pain, the arachidonic acid pathway, apoptosis/angiogenesis, cell adhesion and signal transduction. Compared to placebo, rofecoxib treatment increased the gene expression of ANXA3 (annexin 3), SOD2 (superoxide dismutase 2), SOCS3 (suppressor of cytokine signaling 3) and IL1RN (IL1 receptor antagonist) which are associated with inhibition of phospholipase A(2) and suppression of cytokine signaling cascades, respectively. Both rofecoxib and ibuprofen treatment increased the gene expression of the pro-inflammatory mediators, IL6 and CCL2 (chemokine C-C motif ligand 2), following tissue injury compared to the placebo treatment. These results indicate a complex role for COX-2 in the inflammatory cascade in addition to the well-characterized COX-dependent pathway, as multiple pathways are also involved in rofecoxib-induced anti-inflammatory and analgesic effects at the gene expression level. These findings may also suggest an alternative hypothesis for the adverse effects attributed to selective inhibition of COX-2.

Background: Oral mucositis is the most common sequela of conditioning chemotherapy (CT) for hematopoietic stem cell transplantation (HSCT) and is the principal cause of most of the associated pain. Tumor necrosis factor-alpha (TNF-alpha) is a key pathogenic component of oral mucositis., Objectives: The primary purpose of this study was to describe oral mucositis-related oropharyngeal pain in the setting of HSCT. A secondary purpose was to assess the effectiveness of molecular biology methods for measuring TNF-alpha concentrations in plasma, saliva, and buccal epithelial cells in patients with oral mucositis undergoing HSCT., Methods: This descriptive, correlative study recruited subjects aged >or= 18 years who were scheduled to receive HSCT with CT. Subjects assessed their pain at baseline and 9 days (+/-24 hours) after CT using a pain visual analog scale (VAS) from 0=no pain to 10=worst possible pain, as well as word descriptors of sensory and affective pain. The extent and severity of oral mucositis were evaluated using the Oral Mucositis Assessment Scale. Saliva and blood samples and buccal brush biopsies were obtained at the same time points. Salivary and plasma TNF-alpha concentrations were measured using an enzyme-linked immunosorbent assay. Quantitative real-time polymerase chain reaction testing was used to measure buccal TNF-alpha gene expression. To determine the optimal method of RNA isolation, samples were extracted using 3 different methods: TRIzol, RNeasy, and RLT/TRIzol., Results: Twenty-five adult men and women (mean age, 46 years; age range, 32-68 years; 64% white) underwent HSCT with CT. Significant differences from baseline to day 9 were observed in the severity of oral mucositis (P<0.001), the overall intensity of oral pain (P<0.05), the overall intensity of oral pain with swallowing (P<0.01), the sensory dimension of oral pain with swallowing (P<0.05), and the sensory and affective dimension of oral pain with swallowing (P<0.05). The severity of oral mucositis was significantly associated with the overall intensity of oral pain (P<0.05). Although mean scores for oral pain were low, 8 subjects had clinically unacceptable pain VAS scores (>3) while receiving opioids. Fourteen subjects had measurable increases in buccal TNF-alpha RNA expression at day 9 (P=0.027 vs baseline), as measured using the TRIzol method, which was found to be the best method for measuring this variable. TNF-alpha RNA content in buccal samples was significantly associated with the worst intensity of oral pain with swallowing (partial R(2)=0.19; P<0.05)., Conclusions: Despite the use of opioids, oropharyngeal pain remained a treatment challenge in approximately one third of these subjects after CT with HSCT. The sensitive assay used to measure TNF-alpha gene expression in buccal cells may be useful in investigating molecular events in oral mucositis-related pain, as well as in evaluating the therapeutic response to investigational agents.

Susceptibility to mouse plasmacytomagenesis is a complex genetic trait controlled by several Pctr loci (Pctr1, Pctr2, etc). Congenic strain analysis narrowed the genetic interval surrounding the Pctr2 locus, and genes identified in the interval were sequenced from susceptible BALB/c and resistant DBA/2 mice. Frap (FKBP12 rapamycin-associated protein, mTOR, RAFT) was the only gene differing in amino acid sequence between alleles that correlated with strain sensitivity to tumor development. The in vitro kinase activity of the BALB/c FRAP allele was lower than the DBA/2 allele; phosphorylation of p53 and PHAS1/4EBP1 (properties of heat and acid stability/eukaryotic initiation factor 4E-binding protein) and autophosphorylation of FRAP were less efficient with the BALB/c allele. FRAP also suppressed transformation of NIH 3T3 cells by ras, with DBA/2 FRAP being more efficient than BALB/c FRAP. Rapamycin, a specific inhibitor of FRAP, did not inhibit growth of plasmacytoma cell lines. These studies identify Frap as a candidate tumor suppressor gene, in contrast to many reports that have focused on its prooncogenic properties. Frap may be similar to Tgfb and E2f in exerting both positive and negative growth-regulatory signals, depending on the timing, pathway, or tumor system involved. The failure of rapamycin to inhibit plasma cell tumor growth suggests that FRAP antagonists may not be appropriate for the treatment of plasma cell tumors. Pctr2 joins Pctr1 in possessing alleles that modify susceptibility to plasmacytomagenesis by encoding differences in efficiency of function (efficiency alleles), rather than all-or-none, gain-of-function, or loss-of-function alleles. By analogy, human cancer may also result from the combined effects of several inefficient alleles.

BALB/c mice are susceptible to the development of pristane-induced plasma cell tumors, and have a rare allelic variant in the coding region of the p16(INK4a) (p16) tumor suppressor gene that produces a protein with impaired activity. We have now found that the BALB/c p16 promoter has an allelic variant that may also compromise p16 activity. Following pristane treatment, BALB/c p16 mRNA levels in B cells were lower than that in DBA/2 or C.D2-Pctr1, a resistant BALB/c congenic strain that harbors DBA/2 chromatin surrounding the p16 locus. Four sequence variants were found between BALB/c and DBA/2 in the p16 promoter region. In reporter assays, the DBA promoter was at least four times more active in driving luciferase expression than the BALB/c promoter. Most of the difference in activity was localized to a single nucleotide deletion in BALB/c. This deletion created a consensus binding site for RREB, a ras-responsive transcriptional element with zinc-finger binding motifs. Transient transfections with RREB confirmed that the p16 promoter can be downregulated by RREB, in a Ras- or Mek-dependent manner, and that the BALB/c promoter is more sensitive than DBA/2 to regulation by RREB. BALB/c mice have both regulatory and coding region defects that may contribute to the impairment of p16 gene function.

The plasmacytoma cell line, TEPC 2372, was derived from a malignant plasma cell tumor that developed in the peritoneal cavity of a BALB/c mouse that harbored the transgenic shuttle vector for the assessment of mutagenesis in vivo, lambdaLIZ. TEPC 2372 was found to display the typical features of a BALB/c plasmacytoma. It consisted of pleomorphic plasma cells that secreted a monoclonal immunoglobulin (IgG2b/lambda), was initially dependent on the presence of IL-6 to grow in cell culture, contained a hyperdiploid chromosome complement with a tendency to undergo tetraploidization, and harbored a constitutively active c-myc gene by virtue of a T(6;15) chromosomal translocation. TEPC 2372 was further characterized by the ability to respond to in vitro exposure with 4-NQO (4-nitroquinoline-1-oxide), an oxidative model mutagen, with a vigorous dose-dependent increase in mutagenesis that peaked at a 7.85-fold elevation of mutant rates in lambdaLIZ when compared to background mutant rates in untreated controls. Cotreatment with 4-NQO and BSO (buthionine sulfoximine), a glutathione-depleting compound that causes endogenous oxidative stress, resulted in a 9.03-fold increase in the mutant frequency in lambdaLIZ. These results demonstrated that TEPC 2372, the malignant plasma cell counterpart of the lambdaLIZ-based in vivo mutagenesis assay, may be useful as an in vitro reference point for the further elucidation of oxidative mutagenesis in lymphoid tissues.

The susceptibility of BALB/c mice to pristane-induced plasmacytomas is a complex genetic trait involving multiple loci, while DBA/2 and C57BL/6 strains are genetically resistant to the plasmacytomagenic effects of pristane. In this model system for human B-cell neoplasia, one of the BALB/c susceptibility and modifier loci, Pctr1, was mapped to a 5.7-centimorgan (cM) chromosomal region that included Cdkn2a, which encodes p16(INK4a) and p19(ARF), and the coding sequences for the BALB/c p16(INK4a) and p19(ARF) alleles were found to be polymorphic with respect to their resistant Pctr1 counterparts in DBA/2 and C57BL/6 mice (45). In the present study, alleles of Pctr1, Cdkn2a, and D4Mit15 from a resistant strain (BALB/cDAG) carrying DBA/2 chromatin were introgressively backcrossed to the susceptible BALB/c strain. The resultant C.DAG-Pctr1 Cdkn2a D4Mit15 congenic was more resistant to plasmacytomagenesis than BALB/c, thus narrowing Pctr1 to a 1.5-cM interval. Concomitantly, resistant C57BL/6 mice, from which both gene products of the Cdkn2a gene have been eliminated, developed pristane-induced plasma cell tumors over a shorter latency period than the traditionally susceptible BALB/cAn strain. Biological assays of the p16(INK4a) and p19(ARF) alleles from BALB/c and DBA/2 indicated that the BALB/c p16(INK4a) allele was less active than its DBA/2 counterpart in inducing growth arrest of mouse plasmacytoma cell lines and preventing ras-induced transformation of NIH 3T3 cells, while the two p19(ARF) alleles displayed similar potencies in both assays. We propose that the BALB/c susceptibility/modifier locus, Pctr1, is an "efficiency" allele of the p16(INK4a) gene.

Sera from some patients with polymyositis-scleoderma overlap syndrome (PM-SCL) recognize two antigenically unrelated proteins, PMSCL1 and PMSCL2. Complete mouse Pmscl1 and Pmscl2 cDNA sequences, chromosomal localizations, exon/intron structure, and promoter region sequences of the mouse Pmscl2 gene are presented. The PMSCL1 gene was found to overlap significantly with cyclin A2 in both human and mouse. As such, it may be deduced that PMSCL1 sequences map to human chromosome 4q27 and the proximal portion of mouse chromosome (Chr) 3 where human and mouse cyclin A2 genes reside. Analysis of human and mouse PMSCL1 cDNA sequences provides evidence that the PMSCL1 protein is 68 amino acids longer than previously thought. A BAC containing mouse Pmscl2 was localized to distal mouse Chr 4 by FISH. This BAC contains the microsatellite D4Mit310. D4Mit310 colocalizes with a number of genes that map to human 1p36. In fact, a STS (G25404) located 54.6 cR from the top of human chromosome 1 was found to contain PMSCL2 sequence upon BLAST search., (Copyright 2000 Academic Press.)

Plasma cell tumor induction in mice by pristane is under multigenic control. BALB/c mice are susceptible to tumor development; whereas DBA/2 mice are resistant. Restriction fragment length polymorphisms between BALB/c and DBA/2 for Cdkn2a(p16) and Cdkn2b(p15), and between BALB/c and Mus spretus for Cdkn2c(p18(INK4c)) were used to position these loci with respect to the Pctr1 locus. These cyclin-dependent kinase (CDK) inhibitors mapped to a 6 cM interval of chromosome 4 between Ifna and Tal1. C.D2-Chr 4 congenic strains harboring DBA/2 alleles associated with the Pctr1 locus contained DBA/2 "resistant" alleles of the CDK4/CDK6 inhibitors p16 and p15. On sequencing p16 and p18 cDNAs, two different allelic variants within ankyrin repeat regions of p16 were found between BALB/c and DBA/2 mice. By using an assay involving PCR amplification and restriction enzyme digestion, allelic variants were typed among several inbred strains of mice. One of the variants, G232A, was specific to two inbred strains, BALB/cAn and ABP/Le, of mice and occurred in a highly conserved amino acid in both human and rat p16. When tested with wild-type (DBA/2) p16, both A134C and G232A BALB/c-specific variants of p16 were inefficient in their ability to inhibit the activity of cyclin D2/CDK4 in kinase assays with retinoblastoma protein, suggesting this defective, inherited allele plays an important role in the genetic susceptibility of BALB/c mice for plasmacytoma induction and that p16(INK4a) is a strong candidate for the Pctr1 locus.

The observation that the large ribosomal RNA intron of Tetrahymena is spliced 20-50-fold more rapidly in vivo than in vitro (Brehm SL, Cech TR, 1983, Biochemistry 22:2390-2397; Bass BL, Cech TR, 1984, Nature 308:820-826) suggests facilitation of RNA folding in vivo. To determine whether a specific group I splicing factor is required in Tetrahymena, the intron was inserted into the analogous position of the Escherichia coli 23S rRNA. We report that the intron is rapidly excised from pre-rRNA in bacteria and that the magnitude of the in vivo rate enhancement is similar to that in Tetrahymena. These results demonstrate that a species-specific protein is not required. Instead, a common mechanism of assisting RNA folding is sufficient to accelerate the removal of self-splicing introns from ribosomal RNA.