Macrophage polarization in lymph node granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex.

Tuberculosis in animals is caused by members of the Mycobacterium tuberculosis complex (MTC), with the tuberculous granuloma being the main characteristic lesion. The macrophage is the main cell type involved in the development of the granuloma and presents a wide plasticity ranging from polarization to classically activated or pro-inflammatory macrophages (M1) or to alternatively activated or anti-inflammatory macrophages (M2). Thus, this study aimed to analyze macrophage polarization in granulomas from cattle and pig lymph nodes naturally infected with MTC. Tuberculous granulomas were microscopically categorized into four stages and a panel of myeloid cells (CD172a/calprotectin), M1 macrophage polarization (iNOS/CD68/CD107a), and M2 macrophage polarization (Arg1/CD163) markers were analyzed by immunohistochemistry. CD172a and calprotectin followed the same kinetics, having greater expression in late-stage granulomas in pigs. iNOS and CD68 had higher expression in cattle compared with pigs, and the expression was higher in early-stage granulomas. CD107a immunolabeling was only observed in porcine granulomas, with a higher expression in stage I granulomas. Arg1+ cells were significantly higher in pigs than in cattle, particularly in late-stage granulomas. Quantitative analysis of CD163+ cells showed similar kinetics in both species with a consistent frequency of immunolabeled cells throughout the different stages of the granuloma. Our results indicate that M1 macrophage polarization prevails in cattle during early-stage granulomas (stages I and II), whereas M2 phenotype is observed in later stages. Contrary, and mainly due to the expression of Arg1, M2 macrophage polarization is predominant in pigs in all granuloma stages.

Auritidibacter ignavus in ear discharge of paediatric patients: Two cases reports and literature review.

INTRODUCTION: The development and research of new technologies for identifying microorganisms, has allowed the identification of hitherto unknown bacteria. Auritidibacter ignavus is a newly described Gram-positive rod possibly associated with otitis, although its role as an etiologic agent in otitis is currently controversial. METHODS: We report two cases of recurrent otitis in paediatric patients in which A. ignavus was isolated and review the previous cases reported in the literature. RESULTS: All the isolates were identified as A. ignavus by proteomic and genomic methods. Both patients recovered from their symptoms. CONCLUSION: A. ignavus was recovered from ear discharges of paedriatic patients with chronic ear problems. All the cases previously reported in the literature were adults. More evidence is needed for the association between A. ignavus and otitis, since data regarding this species are still scarce.

Demand charges reduction with photovoltaics in industry.

Demand charges are widely used for commercial and industrial consumers. These costs are often not well known, let alone the effects that PV can have on them. This work proposes a methodology to assess the effect of PV on reducing these charges and to optimise the power to be contracted, using techniques taken from exploratory data analysis. This methodology is applied to five case studies of industrial consumers from different sectors in Spain, finding savings between 5 % and 11 % of demand charges in industries with continuous operation and up to 28 % in cases of discontinuous operation. These savings can be even greater if the maximum power that can be contracted is lower than the optimum. The demand charges in Spain consist of a fixed part proportional to the contracted power and a variable part depending on the power peaks exceeding it. Since for the variable part the coincident and non-coincident models coexist, a comparison is made between the two models, finding that in the general case PV users can achieve higher savings with the coincident model.

Retrospective evaluation of hematological ratios in canine parvovirosis: 401 cases.

BACKGROUND: The utility of neutrophil-to-lymphocyte ratio (NLR), platelet to-lymphocyte ratio (PLR) and monocyte-to-lymphocyte ratio (MLR) as prognostic indicators has not been investigated in canine parvovirosis (CPV). HYPOTHESIS: To evaluate whether these hematological ratios obtained at hospital admission in CPV are associated with outcome or duration of hospitalization. ANIMALS: Four hundred one client-owned dogs presented with CPV. Methods-Retrospective multicenter cohort study. Medical records were reviewed to identify dogs with CPV. Data regarding signalment, complete blood count at admission, duration of hospitalization and outcome were collected. RESULTS: Of the 401 dogs included in the study, 336 (83.8%) survived to discharge. The median (25th and 75th percentiles) PLR in nonsurvivors (336.56 [159.84-635.77]) was significantly higher than in survivors (217.65 [117.67-389.65]) (P = .003). The area under the receiver-operating characteristic curve for nonsurvival was 0.615 (95% CI [0.593-0.691], P = .003). A cut off of 700 showed a 21.5% sensitivity and 90% specificity for nonsurvival. No association was observed between hospitalization duration and either hematological ratios or total WBC counts. The median (25th and 75th percentiles) lymphocyte count was below reference interval in all dogs and was significantly lower in the dogs which died (0.82 × 109 /L [0.5-1.87]) than in survivors (1.27 × 109 /L [0.73-2.22]) (P = .005). The median (25th and 75th percentiles) monocyte count however was lower in survivors (0.38 × 109 /L [0.29-1.59]), than in nonsurvivors (0.73 × 109 /L [0.1-2]) (P = .002). CONCLUSIONS: Evaluation of PLR at hospital admission might be a useful marker of disease severity and could have prognostic value in dogs with CPV.

FOVOCIP study: a multicenter randomized trial of fosfomycin versus ciprofloxacin for febrile neutropenia in hematologic patients-efficacy and microbiologic safety.

BACKGROUND: Multidrug-resistant Gram-negative bacterial (MRGNB) infections represent a major public health threat. Cancer patients and, among them, hematological patients are most vulnerable to these infections. Gut colonization by MRGNB is a common phenomenon occurring during hospitalization and chemotherapy exposure. In the neutropenic phase that occurs after chemotherapy, MRGNB translocation occurs increasing patient's mortality. Fluoroquinolone prophylaxis with ciprofloxacin or levofloxacin efficacy is now being questioned due to the increase of incidence in MRGNB. METHODS: A phase III randomized, controlled, clinical trial, open-label parallel-group with a 1:1 ratio, aimed to demonstrate the non-inferiority of oral fosfomycin versus oral ciprofloxacin for febrile neutropenia prevention in patients with acute leukemia (AL) or hematopoietic cell transplant (HSC) receptors. Weekly surveillance cultures are planned to detect gut colonization. Changes in fecal microbiome at the beginning and end of prophylaxis will also be analyzed. DISCUSSION: This trial will provide evidence of the efficacy of an alternative drug to ciprofloxacin for febrile neutropenia prevention in high-risk hematological patients. The battery of planned microbiological studies will allow us to evaluate prospectively the microbiological safety of both pharmacological strategies in terms of the selection of MRGNB occurring in each arm. In addition, valuable information on the way in which each drug changes the fecal microbiome of the patients throughout the treatment will be generated. TRIAL REGISTRATION: Clinical trials NCT05311254, Registered on 5 April 2022, https://clinicaltrials.gov/ct2/show/NCT05311254?term=FOVOCIP&cntry=ES&draw=2&rank=1 . PROTOCOL VERSION: 3.0, dated 20 May 2022.

Porcine Macrophage Markers and Populations: An Update.

Besides its importance as a livestock species, pig is increasingly being used as an animal model for biomedical research. Macrophages play critical roles in immunity to pathogens, tissue development, homeostasis and tissue repair. These cells are also primary targets for replication of viruses such as African swine fever virus, classical swine fever virus, and porcine respiratory and reproductive syndrome virus, which can cause huge economic losses to the pig industry. In this article, we review the current status of knowledge on porcine macrophages, starting by reviewing the markers available for their phenotypical characterization and following with the characteristics of the main macrophage populations described in different organs, as well as the effect of polarization conditions on their phenotype and function. We will also review available cell lines suitable for studies on the biology of porcine macrophages and their interaction with pathogens.

Transferring Substituents from Alkynes to Furans and Pyrroles through Heteronorbornadienes as Intermediates: Synthesis of ß-Substituted Pyrroles/Furans.

The use of 7-oxa/azanorbornadienes as synthetic intermediates for the preparation of 3/4-substituted (ß-substituted) furans/pyrroles is presented. The method lies in the inverse electron demand Diels-Alder (iEDDA) cycloaddition between a substituted heteronorbornadiene and an electron-poor tetrazine followed by spontaneous fragmentation of the resulting cycloadduct via two retro-Diels-Alder (rDA) reactions affording a ß-substituted furan/pyrrole. The scope of this tandem iEDDA/rDA/rDA reaction was explored in the preparation of 29 heterocycles. A one-pot procedure starting directly from the alkyne precursors of the heteronorbornadiene intermediates is also described.

Identification and biotechnological characterisation of yeast microbiota involved in spontaneous fermented wholegrain sourdoughs.

BACKGROUND: New strategies in the cereal-based industry has brought about the elaboration of new sourdoughs with better microbial stability and safety as well as nutritional value such as those based on wholegrain flours. This has led to an increasing interest in the selection of adapted yeasts for using them as new starters. Therefore, this study aimed to isolate, identify, and characterise diverse yeast strains from wholegrain spontaneous sourdoughs. RESULTS: Three wholegrain sourdoughs (wheat, rye, and oat) were fermented and monitored for 96 h. Minimum pH values ranged from 3.1 to 3.5 while maximum yeast counts were reached at 72 h. A total of 76 yeast isolates were identified by polymerase chain reaction random amplification of polymorphic DNA (PCR-RAPD) and catalogued in six different species by sequencing the internal transcribed spacer (ITS) region. The major species were Candida glabrata, Saccharomyces cerevisiae, Kazachstania unispora, and Wickerhamomyces anomalus. The studied kinetic parameters of the growth curves (λ, G, ODmax , and µmax ) and the fermentation capacity allowed to ascertain that 12 and 5 strains, respectively, were better than baker's yeast control. The fibre assimilation ability (cellulose, xylose, and ß-glucan) was observed in the 27% of the strains and only four strains showed phytase activity. CONCLUSIONS: The yeast population in the three wholegrain sourdoughs were variable along the fermentation time. Genetic identification showed that strains and species presented a different trend for each sourdough although common species were determined (e.g., W. anomalus). Candida glabrata (4T1) and Saccharomyces cerevisiae (3A6) showed, respectively, better kinetics and impedance results than the positive control, while W. anomalus (C4) was notorious in fibre assimilation and phytase degradation. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Microascus gracilis y otomicosis

Microascus spp, teleomorfo de Scopulariopsis, es un hongo saprofito encontrado normalmente en suelo, alimentos, vegetales e incluso en ambientes interiores. Considerado un contaminante ambiental, se caracteriza por la resistencia intrínseca a los antifúngicos disponibles. Existen escasas referencias de infecciones por Microascus gracilis, asociándose como causa de eumicetoma o enfermedad diseminada en pacientes sometidos a trasplante pulmonar. Presentamos un caso de otomicosis por M. gracilis con el fin de considerar la búsqueda de hongos en los cultivos óticos y poner de relevancia el poder patógeno y colonizador de este agente.

Microascus spp, a teleomorph of Scopulariopsis, is a saprophytic fungus normally found in soil, food, vegetables, and even indoors. Considered an environmental pollutant, it is characterized by its intrinsic resistance to available antifungals. There are few references to infections by Microascus gracilis, associating it as a cause of eumycetoma or disseminated disease in lung transplant recipients. We present a case of otomycosis caused by M. gracilis, to consider the search for fungi in ear cultures and highlight the pathogenic and colonizing power of this agent.

Molecular characterization of epithelial-mesenchymal transition and medical treatment related-genes in non-functioning pituitary neuroendocrine tumors.

Introduction: Different medical therapies have been developed for pituitary adenomas. However, Non-Functioning Pituitary Neuroendocrine Tumors (NF-PitNET) have shown little response to them. Furthermore, epithelial-mesenchymal transition (EMT) has been linked to resistance to medical treatment in a significant number of tumors, including pituitary adenomas. Methods: We aimed to evaluate the expression of EMT-related markers in 72 NF-PitNET and 16 non-tumoral pituitaries. To further explore the potential usefulness of medical treatment for NF-PitNET we assessed the expression of somatostatin receptors and dopamine-associated genes. Results: We found that SNAI1, SNAI2, Vimentin, KLK10, PEBP1, Ki-67 and SSTR2 were associated with invasive NF-PitNET. Furthermore, we found that the EMT phenomenon was more common in NF-PitNET than in GH-secreting pituitary tumors. Interestingly, PEBP1 was overexpressed in recurrent NF-PitNET, and could predict growth recurrence with 100% sensitivity but only 43% specificity. In parallel with previously reported studies, SSTR3 is highly expressed in our NF-PitNET cohort. However, SSTR3 expression is highly heterogeneous among the different histological variants of NF-PitNET with very low levels in silent corticotroph adenomas. Conclusion: NF-PitNET showed an enhanced EMT phenomenon. SSTR3 targeting could be a good therapeutic candidate in NF-PitNET except for silent corticotroph adenomas, which express very low levels of this receptor. In addition, PEBP1 could be an informative biomarker of tumor regrowth, useful for predictive medicine in NF-PitNET.

Whole genome analysis of Gram-negative bacteria using the EPISEQ CS application and other bioinformatic platforms.

OBJECTIVES: To determine genomic characteristics and molecular epidemiology of carbapenem non-susceptible Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa from medical centres of Mexico using whole genome sequencing data analysed with the EPISEQⓇ CS application and other bioinformatic platforms. METHODS: Clinical isolates collected from 28 centres in Mexico included carbapenem-non-susceptible K. pneumoniae (n = 22), E. coli (n = 24), A. baumannii (n = 16), and P. aeruginosa (n = 13). Isolates were subjected to whole genome sequencing using the Illumina (MiSeq) platform. FASTQ files were uploaded to the EPISEQⓇ CS application for analysis. Additionally, the tools Kleborate v2.0.4 and Pathogenwatch were used as comparators for Klebsiella genomes, and the bacterial whole genome sequence typing database was used for E. coli and A. baumannii. RESULTS: For K. pneumoniae, both bioinformatic approaches detected multiple genes encoding aminoglycoside, quinolone, and phenicol resistance, and the presence of blaNDM-1 explained carbapenem non-susceptibility in 18 strains and blaKPC-3 in four strains. Regarding E. coli, both EPISEQⓇ CS and bacterial whole genome sequence typing database analyses detected multiple virulence and resistance genes: 20 of 24 (83.3%) strains carried blaNDM, 3 of 24 (12.4%) carried blaOXA-232, and 1 carried blaOXA-181. Genes that confer resistance to aminoglycosides, tetracyclines, sulfonamides, phenicols, trimethoprim, and macrolides were also detected by both platforms. Regarding A. baumannii, the most frequent carbapenemase-encoding gene detected by both platforms was blaOXA-72, followed by blaOXA-66. Both approaches detected similar genes for aminoglycosides, carbapenems, tetracyclines, phenicols, and sulfonamides. Regarding P. aeruginosa, blaVIM, blaIMP, and blaGES were the more frequently detected. Multiple virulence genes were detected in all strains. CONCLUSION: Compared to the other available platforms, EPISEQⓇ CS enabled a comprehensive resistance and virulence analysis, providing a reliable method for bacterial strain typing and characterization of the virulome and resistome.

Nefrolitotomía Endoscópica ex vivo del injerto renal de donante vivo con instrumento flexible

Introduction: The finding of an asymptomatic stone in the study of a living kidney donor does not necessarily contraindicate donation, however, there is no consensus on the management of these cases. The use of a graft with lithiasis may represent a risk of recurrence in the remaining kidney in the donor and eventual obstructive complications in the transplanted kidney. The objective of this work is to present the usefulness of ureteroscopy (URS) to resolve lithiasis ex vivo before transplantation. Material and Methods: Donors with a small, asymptomatic kidney stone and with an analysis of lithogenic factors without relevant findings were considered to continue in the donation process. The kidney unit with stone was selected for nephrectomy. Results: Four donor kidneys underwent flexible URS after nephrectomy under hypothermic preservation conditions during bench preparation. The average time of the procedure was 35 minutes and the stone was extracted in all cases without incident. The transplant was carried out in the usual way and the evolution of the recipients was without complications and with excellent renal function. During follow-up, no recurrence of lithiasis was observed in donors or recipients. Conclusions: In this experience, the URS of the donor kidney was a feasible procedure and was not associated with adverse consequences for the graft. The main advantage of this procedure is to avoid the potential risk to the recipient of an obstructive graft complication.

Clostridium paraputrificum bacteremia in a 64-year-old woman with colon carcinoma.

Here we report a case of bacteremia caused by Clostridium paraputrificum in a 64-year-old woman with colon carcinoma and gastrointestinal disease. Using the new EUCAST 2022 clinical breakpoints for Clostridium perfringens, the isolate was susceptible to metronidazole and vancomycin, but resistant to benzylpenicillin, meropenem, and clindamycin. Thus, treatment with metronidazole should be considered in all patients with Clostridium bacteremia until antibiotic susceptibility is determined to minimize the risk of treatment failure.

Recommendations of the Spanish Antibiogram Committee (COESANT) for in vitro susceptibility testing of antimicrobial agents by disk diffusion.

Disk diffusion is a well standardized method that provides reliable categorical results to guide antimicrobial therapy in numerous types of infections. Based on the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which are widely implemented in Spain, the Spanish Antibiogram Committee (COESANT) has drawn up recommendations for antimicrobial selection by the disk diffusion technique, including selective reporting and its use for the detection of resistance mechanisms. Factors affecting disk diffusion results, along with advantages and shortcomings of the method, are also discussed.

Quantitative analysis of MGMT promoter methylation status changes by pyrosequencing in recurrent glioblastoma.

MGMT promoter methylation status can change in response to several factors, treatment with alkylating therapy being the mechanism more commonly cited in the literature. Some authors have attempted to quantify these alterations, with inconsistent results. This study aims to determine changes in MGMT promoter methylation status by pyrosequencing, which quantitatively yields results, in a cohort of patients reoperated for recurrent glioblastoma and having previously completed the Stupp protocol. Methylation status of the MGMT promoter gene of a total of 24 pairs of glioblastoma preselected tumor samples was retrospectively analyzed using pyrosequencing and depicted as percentages or categories (hypermethylated, intermediate methylation, unmethylated). Matched samples were compared using Wilcoxon signed-rank test, and log-rank test was used to establish a correlation with survival data. The median value of MGMT promoter methylation status declined after adjuvant treatment from 20.35% to 14.25% (p = 0.346). A significant correlation between methylation in primary samples and overall survival (p = 0.05) and progression-free survival (p = 0.024) was found. Intermediate methylation status at recurrence was linked to greater survival after progression, without reaching statistical significance (post-progression survival [PPS]) (p = 0.217). Although treatment with alkylating chemotherapy was a common feature in all patients of our cohort, switching in both directions was observed when MGMT promoter methylation status was analyzed as a continuous variable. These data suggest that the dynamics of epigenetics may be very complex and not entirely explained by clonal selection influenced by temozolomide.

Spanish Antibiogram Committee (COESANT) recommendations for cumulative antibiogram reports.

The Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) presents in this document a series of recommendations intending to unify how cumulative antibiogram reports must be made in Clinical Microbiology Spanish laboratories. This article is based on the information included in the Clinical Microbiology Procedure No. 51, «Preparation of cumulative reports on antimicrobial susceptibility¼ of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), published in 2014. The recommendations also include the modifications in the definition of clinical interpretive categories recently published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2019. Its final objective is to establish a homogeneous way of preparing these summaries to compare results from different centers or aggregate the information from these in order to carry out an adequate local or even national surveillance regarding the evolution of antimicrobial susceptibility.

[Living-donor kidney graft ex vivo endoscopic nephrolithotomy with flexible instrument]. / Nefrolitotomía Endoscópica ex vivo del injerto renal de donante vivo con instrumento flexible.

INTRODUCTION: The finding of an asymptomatic stone in the study of a living kidney donor does not necessarily contraindicate donation, however, there is no consensus on the management of these cases. The use of a graft with lithiasis may represent a risk of recurrence in the remaining kidney in the donor and eventual obstructive complications in the transplanted kidney. The objective of this work is to present the usefulness of ureteroscopy (URS) to resolve lithiasis ex vivo before transplantation. MATERIAL AND METHODS: Donors with a small, asymptomatic kidney stone and with an analysis of lithogenic factors without relevant findings were considered to continue in the donation process. The kidney unit with stone was selected for nephrectomy. RESULTS: Four donor kidneys underwent flexible URS after nephrectomy under hypothermic preservation conditions during bench preparation. The average time of the procedure was 35 minutes and the stone was extracted in all cases without incident. The transplant was carried out in the usual way and the evolution of the recipients was without complications and with excellent renal function. During follow-up, no recurrence of lithiasis was observed in donors or recipients. CONCLUSIONS: In this experience, the URS of the donor kidney was a feasible procedure and was not associated with adverse consequences for the graft. The main advantage of this procedure is to avoid the potential risk to the recipient of an obstructive graft complication.

Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs.

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1ß, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1ß in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1ß, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1ß and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1ß positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1ß at the site of inflammation during the inflammatory process.

Predicting malignancy in small testicular lesions.

Introduction: Small testicular lesions ≤20 mm (STL) detected by ultrasound (US), usually non-palpable, have been reported to be benign in up to 80% of cases. Thus, partial orchiectomy with or without frozen section examination and surveillance has been advocated for these kinds of lesions. We seek to report the proportion of benign lesions in testicular tumors ≤20 mm detected by US in our population and explore the predicting factors of malignancy. Material and methods: A retrospective descriptive study of orchiectomies performed for testicular tumors in patients older than 15 years between 2005 and 2019 was performed, including all patients with lesions ≤20 mm on US imaging. Results: A total of 70 patients with STL were included (mean age 34.6 ±10.8 years). Overall, 69% of the lesions were malignant while the smallest lesions (≤10 mm) showed 61% of cancer. Moreover, in the subgroup of non-palpable lesions ≤10 mm, 50% were malignant. Multifocal tumors were found in 18 subjects with a malignancy rate of 88%. There was a significant association between maximum size on US, multifocality and malignancy. Neither tumor markers nor palpability foretold a malignant lesion. A predictive model including size and multifocality was created showing a positive predictive value of 83.3%. Conclusions: US maximum size and multifocality were predictors of malignancy in STL. However, even the smallest lesions showed a 50% chance of being malignant, thus surgery with or without intraoperative biopsy is warranted in most cases.

CD9 expression in porcine blood CD4+ T cells delineates two subsets with phenotypic characteristics of central and effector memory cells.

In this report, we describe the characterization of a new monoclonal antibody, named 4H5CR4, against porcine CD9. Its use in combination with antibodies to CD4, CD8α, and 2E3 allows to distinguish at least five main CD4+ T cell subsets. Analysis on these subsets of CD45RA, CD27, CD29, CD95, CCR7, and SLA-DR markers depicts a progressive model of CD4+ T cell development. CD4+ 2E3+ CD8α- CD9- cells are the least differentiated population of naïve cells, whereas the CD4+ 2E3- CD8α+CD9+ and CD4+ 2E3- CD8α+ CD9- cells display phenotypic features of central and effector memory T helper cells, respectively. The latter subsets were able to produce IFN-γ after polyclonal activation with PMA/Ionomycin; however, in vitro virus-specific IFN-γ production of PBMCs collected at 38-44 days after pseudorabies virus vaccination was dominated by cells with a CD9+ phenotype. Therefore, CD9 appears to be a useful marker to investigate CD4+ T cell heterogeneity in swine.