Unmasking Budgerigar Splenic Leukocyte Populations With Single‐Cell Transcriptomics and Multiplex RNA In Situ Hybridization

Primary central nervous system lymphomas (PCNSL) are aggressive extranodal malignancies confined to the central nervous system (CNS), mostly of diffuse large B cell histotype. Despite improved understanding of the malignant B cells, little is known on the tumor microenvironment and on the response of the adaptive immunity against PCNSL. The majority of B-cell lymphomas, including PCNSL originate from germinal center (GC) B cells. The GC is the main source of memory B cells and plasma cell generation, which produce high affinity antibodies and are necessary to protect us against invading microorganisms. However, the beneficial role of GC B cells in immunity is counterbalanced by their detrimental role in lymphomagenesis. Germinal center B cells express a distinct set of chemokine receptors, which regulate their migration and positioning during and after germinal center reaction. Similar to centrocytes and centroblasts, malignant B cells derived from germinal centers can retain a particular set of chemokine receptors, which allows them to respond to their cognate ligands expressed in the microenvironment. Therefore investigation of lymphocyte chemoattractants in secondary lymphoid organs as well as in extranodal lymphomas does not only improve our understanding of B cell trafficking within secondary lymphoid organs, but also helps us understanding tumor cell distribution and dissemination of malignant B cells as well as tumor infiltrating lymphocytes. Gene expression analysis has previously shown, that malignant B cells in PCNSL resemble late germinal center B cells and express classical B cell chemokine receptors. This work focuses on the chemokine microenvironment and the potential role of bystander cells in PCNSL and their effects on malignant B cells as well as tumor infiltrating lymphocytes. The project includes four major sections: 1. Analysis of the expression of B cell attracting chemokines under normal and inflammatory conditions in human secondary lymphoid organs. 2. Analysis of T and B cell attracting chemokines in extranodal CNS lymphomas, 3. Analysis of type, density and localization of tumor infiltrating lymphocytes in PCNSL, 4. Analysis of the effect of coexpressed chemokines in PCNSL on the migratory responses of tumor-infiltrating lymphocytes and malignant B cells. We show by immunhistochemistry and in-situ hybridization, that there is a specific expression pattern of homeostatically expressed chemokines CXCL12, CXCL13 and CCL21 in normal, non-inflamed human secondary lymphoid organs. Under inflammatory conditions, the expression pattern of macrophage- derived CXCL12 and follicular dendritic cell- derived CXCL13 within the germinal center changes significantly. Within the germinal center, macrophage-derived CXCL12 and follicular dendritic cell-derived CXCL13 build a meshwork in which germinal center B cells reside. While CXCL13 shows a clear gradient between the dark and the light zone of the germinal center in human secondary lymphoid tissue, we could not detect a clear gradient for CXCL12 between the two zones. Analysis of CXCL12 and CXCL13 in primary central nervous system lymphoma showed an expression pattern similar to the one in germinal centers of secondary lymphoid organs. In addition, we identified a fraction of CXCL13-expressing lymphocytes in PCNSL. CXCL13-expression is a hallmark of germinal center T cells known as follicular T helper cells (TFH), which provide help to germinal center B cells. Yet, the majority of tumor infiltrating lymphocytes in PCNSL are CD8+ T cells, which show Granzyme B activity and vigorous proliferation. Tumor infiltrating CD8+ T cells show a higher frequency in the perivascular areas of small and intermediate vessels in PCNSL. They accumulate in areas with high expression of the inflammatory chemokine CXCL9. Perivascular CXCL9 is upregulated by perivascular macrophages and pericytes under inflammatory conditions in the CNS, indicating an important role for pericytes and perivascular macrophages in the recruitment of tumor infiltrating lymphocytes. Moreover, CXCL9 and CXCL12 are coexpressed on the tumor vasculature within PCNSL and can form heterocomplexes. Our in-vitro experiments show, that in the presence of CXCL9, CXCL12-induced migration is enhanced not only on CXCR4+/CXCR3+/CD8+ T cells but also on CXCR4+/CXCR3- malignant B cells. Our findings indicate, that malignant B cells in PCNSL may encounter a germinal-center like chemokine environment, which traps malignant B cells within the CNS. In addition, our results reveal the presence of a strong chemoattractant stimulus in the perivascular microenvironment, which might serve as regulator for the recruitment of tumor infiltrating lymphocytes and for the angiocentric positioning of malignant B cells in the perivascular cuff.

BackgroundHigh-risk human papillomavirus (HR-HPV) is notoriously associated with tumor progression in a broad spectrum of malignancies. Detection of HR-HPV is clinically important in the management of HPV-related carcinomas, particularly in cervical cancer and oropharyngeal squamous cell carcinoma (OPSCC). Several methods for HPV detection are currently available including Polymerase chain reaction (PCR)-based techniques, DNA in situ hybridization (ISH), RNA ISH, and p16 immunohistochemistry (IHC). Currently, the guidelines for HPV detection in cervical carcinoma are available, while no clear consensus has not yet been reached on the gold standard for HPV testing in OPSCC. Multimodality testing could help to reliably identify patients with transcriptionally active high-risk HPV-positive.MethodsWe propose a multiplex approach carrying out HPV RNA ISH and p16 IHC on the same slide to detect simultaneously HPV E6/E7 transcripts and p16INK4a overexpression. We tested this assay in two different series one of the cervical cancers with p16-positive, as control, and the other of oropharyngeal squamous cell carcinomas with blind p16 status.ResultsThe multiplex HPV RNA ISH /p16 IHC results in the series both of the cervical cancers and the oral-oropharyngeal cancers were fully concordant with the previous results achieved through the classic p16 IHC and HPV RNA scope carried out on two different slides.ConclusionsOur results suggesting several advantages of this technical approach, namely an easy interpretation fully in the light field, the feasibility in formalin-fixed paraffin-embedded tissue sections, complete automation and a potential wide spreadable for routine testing in several clinical laboratories.

miRNA Profiling of B Cell Subsets: Specific miRNA Profile for Germinal Center B Cells with a Marked Variation Between Centroblast and Centrocytes.

This chapter discussed the important differences in the anatomy and physiology of some of the known species of birds, all belonging to the class Aves. The possession of feathers, and in the majority of species the ability to fly, is the reason for their success. Flight allows them to colonize new habitats, find new sources of food and escape predators. Much of their anatomy and physiology has evolved to facilitate life in the air. The most commonly kept species of cage and aviary birds include several members of the order Psittaciformes such as the budgerigar (Melopsittacus undulatus), the cockatiel (Nymphicus hollandicus) and several species of parrot. These birds are brightly coloured and have an ability to mimic sounds, giving rise to their apparent ability to 'talk'. Many species of perching bird from the order Passeriformes are also popular in aviaries. These include finches such as the Zebra Finch (Taeniopygia poephilia guttata) and canaries (Canarius serinus). Highlights of this chapter includes examples of the way in which the bird is adapted for flight, important parts of the avian digestive system (tongue, rectum, crop, beak, gizzard, cloaca, ileum, caecum, duodenum, oesophagus, proventriculus, and large intestine), the functions of the female reproductive system which includes the infundibulum, magnum, isthmus, shell gland, and vagina, and details on the passage of air through the respiratory system of the bird, from inspiration to eventual expiration.

Extensive burn injury (>30% total body surface area [TBSA]) leads to significant inflammation and associated organ damage. The spleen is a major reservoir of lymphocytes which decrease systemically post-burn. Abnormal splenic function may relate to the inflammatory response after burn. While intravenous (IV) fluid resuscitation is the current standard for burn care, enteral resuscitation has also shown promise, and may be leveraged to help splenic recovery. However, the effect of resuscitation on the spleen post-burn is largely unstudied. Thirty anesthetized Yorkshire swine subjected to 40% TBSA contact burns were randomized to one of five groups (n=6/group); no fluids, ad lib water, volume-matched Oral Rehydration Salts (ORS) from the World Health Organization, limited-volume (15mL/kg/d) ORS, or IV lactated Ringer’s solution at 2mL/kg/%TBSA/d (IV). Computerized tomography (CT) scans were performed before and 48 h post-burn, at which point spleens were harvested. Histology was performed to localize splenic CD3 and measure the proportion of red and white pulp. Gene and protein expression was analyzed via qPCR, Western blot, and multiplex. The splenic artery diameter was maintained with water (-0.2 ± 0.32mm), limited-volume (0.36 ± 0.37mm), or volume-matched ORS (-0.2 ± 0.04mm), while no fluids led to a reduction (-0.97 ± 0.14mm) and IV fluids led to dilation (0.68 ± 0.30mm) 48 h post-burn. However, no differences in spleen wet-to-dry ratios were detected among treatments. Levels of white pulp were highest in the ad lib water group (233.0 ± 1.7AU), volume-matched ORS (229.7 ± 2.5), and IV groups (233.3 ± 1.363) compared to no fluids (221.9 ± 5.2) or low-volume ORS (218.7 ± 3.4). Gene expression of complement C5 and uteroglobin-related protein 2 were upregulated in IV animals by 2.76 and 2.43 fold, respectively, when compared to volume matched ORS. Protein expression of CD3 tended to be greatest in animals receiving ORS, and IV fluids. Protein levels of IL1ra were greatest in low and volume matched ORS compared to other groups. Burn injury leads to significant changes in splenic leukocytes, which can be altered with different resuscitation strategies. Specifically, access to enteral fluids preserves splenic lymphocytes post-burn similarly to IV fluids, and may uniquely alter the inflammatory response. Enteral fluids also maintain splenic perfusion, with minimal change in splenic artery diameter. The therapeutic efficacy of enteral resuscitation in burn injury warrants further investigation. Enteral fluids represent a viable means of maintaining splenic perfusion which could easily be used to buy time in mass casualty and prolonged field care scenarios in patients with no concomitant abdominal injuries.

BackgroundThe high-risk human papillomavirus (HPV) infection represents one of the main etiologic pathways of penile carcinogenesis in approximately 30–50 % of cases. Several techniques for the detection of HPV are currently available including Polymerase chain reaction-based techniques, DNA and RNA in situ hybridization (ISH), p16 immunohistochemistry (IHC). The multiplex HPV RNA ISH/p16 IHC is a novel technique for the simultaneous detection of HPV E6/E7 transcripts and p16INK4a overexpression on the same slide in a single assay. The main aim of this study was to evaluate the discrepancy of p16 IHC expression relatively to HPV RNA ISH in penile cancer tissue.MethodsWe collected a series of 60 PCs. HPV has been analysed through the RNA ISH, p16 IHC and the multiplex HPV RNA ISH/p16 IHC.ResultsThe multiplex HPV RNA ISH /p16 IHC results in the series were in complete agreement with the previous results obtained through the classic p16 IHC and HPV RNA scope carried out on two different slides. The multiplex HPV RNA ISH /p16 IHC showed that HPV positivity in our series is more frequently in usual squamous cell carcinoma than in special histotypes (19 out of 60 − 15 %- versus 6 out of 60 − 10 %-), in high-grade than in moderate/low grade carcinomas (6 out of 60 − 10 %- versus 4 out of 60 − 6.7 %-). In addition, our data revealed that in 5 out of 20 cases with p16 high intensity expression is not associated with HPV RNA ISH positivity.ConclusionsOur findings emphasize that the use of p16 as a surrogate of HPV positivity was unsuccessful in approximatively 8 % of cases analysed in our series. Indeed, p16 IHC showed a sensitivity of 100 % and a specificity of 71 %, with a positive predictive value (PPV) of 54 % and a negative predictive value of 100 %; when considering high intensity, p16 IHC showed a sensitivity of 100 %, a specificity of 89 %, with a PPV of 75 % and NPV of 100 %.Since HPV positivity could represent a relevant prognostic and predictive value, the correct characterization offered by this approach appears to be of paramount importance.

Three out of four emerging infectious diseases of humans originated in animals. The COVID‐19 pandemic highlights the importance of understanding how the immune systems of animals interact with viruses, bacteria, and parasites allowing them to serve as disease reservoirs. Unfortunately, the tools to study the immune system of most animal species are not at the level needed to investigate these interactions. Therefore, our laboratory is constructing a new reporter plasmid that will allow for the development of species‐specific transcription factor‐activated reporter cell lines. The initial proof of concept is focused on avian species. Birds are known reservoirs for zoonotic pathogens like influenza A viruses, West Nile virus, Salmonella, Campylobacter, among others. Understanding how the avian immune system responds to zoonotic pathogens is critical to understanding how they serve as reservoirs, and potentially developing novel ways to prevent disease in humans. Transcription factors regulating pro‐ and anti‐inflammatory genes were chosen and candidate binding sites conserved across multiple avian species were identified. These candidate transcriptional binding sites were inserted into a novel dual reporter plasmid and screened for their ability to control expression of green fluorescent protein. Plasmids containing validated binding sites will then be stably integrated into the genomes of different avian cell lines generating a transcription factor‐specific reporter cell line. These cell lines will then be used as a library to characterize the transcriptional response of various cell lineages following stimulation by different pathogens, pathogen pattern molecular patterns, and cytokines. Ultimately this platform will be expanded to other species, allowing us to compare the in vitro host response across different mammalian, avian, and teleost species. Shedding new light on the role the immune response plays in the pathogenicity of different zoonotic agents.

Germinal centers

Undertail body wall herniation involving the oviduct in a budgerigar (Melopsittacus undulatus)

ABSTRACTAccumulating comparative and interdisciplinary research supports a brain cooling function to yawning. In particular, previous research has shown significant decreases in both brain and skull temperature following yawning in mammals. In a recent study using a thermal imaging camera, significant reductions in both the cornea and concha temperature were observed following yawns in the high-yawning subline of Sprague-Dawley rats. Here, we performed a similar experiment to investigate shifts in facial temperature surrounding yawning in an avian species with more typical yawning patterns: budgerigars (Melopsittacus undulatus). In particular, we took maximal surface temperature recordings from the face (cere or eye) from 13 birds over a one-hour period to track changes before and after yawns. Similar to previous findings in high-yawning rats, we identified significant cooling (−0.36°C) of the face 10–20 seconds following yawning in budgerigars. Consistent with the hypothesis that yawns serve a thermoregulatory function, facial temperatures were slightly elevated just prior to yawning and then decreased significantly below baseline levels immediately thereafter. Similarly, birds that yawned during the trials had consistently higher facial temperatures compared to those that did not yawn. Moreover, yawn latency and overall yawn frequency were strongly correlated with the highest facial temperature recorded from each bird across trials. These results provide convergent evidence in support of a brain cooling function to yawning, and further validate the use of thermal imaging to monitor changes in skull temperature surrounding yawning events.

Perception of faces by 4 budgerigars (Melopsittacus undulatus), a species of small parrot, was studied with a same-different discrimination task. Reaction times were taken as a measure of the similarity between pairs of faces and analyzed with multidimensional scaling to reveal patterns of similarity among the faces. The perception of natural faces was tested to determine which characteristics were perceptually salient. Color, patterns of markings, darkness of the iris, and size of the pupil corresponded to the observed patterns of similarity among the faces. Differences among budgerigar faces were more salient than differences among zebra finch faces, and budgerigar faces were perceptually distinct from the faces of other avian species. The results from these experiments provide a basis for understanding the ways in which these signals function in the coordination of social behaviors.

Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

The brain of the budgerigar (Melopsittacus undulatus), a small parrot that acquires new vocalizations throughout life, was examined for immunoreactivity to the opioid peptide methionine enkephalin (mENK). mENK is a highly prominent feature of the chemical architecture of the forebrain vocal system of oscine songbirds. Forebrain vocal control nuclei are believed to have evolved independently in parrots and songbirds (Streidter [1994] J. Comp. Neurol. 343:35-56); however, recent studies have found similarities in the neural organization of vocal control pathways in budgerigars and songbirds (Durand et al. [1997] J. Comp. Neurol. 377:179-206). Among the similarities are the existence of recursive pathways interconnecting vocal control neurons in the archistriatum, basal ganglia (i.e., lobus parolfactorius), and dorsal thalamus. In the present study, we found that all vocal control nuclei within the budgerigar forebrain exhibit prominent mENK-like immunoreactivity (ELI) in fibers and somata. We also found striking similarities between the morphology of ELI elements in budgerigar vocal control nuclei and that described previously in songbird vocal nuclei. Despite these similarities, the budgerigar dorsal striatopallidum (lobus parolfactorius, paleostriatum augmentatum, and paleostriatum primitivum) and somatomotor (anterior) archistriatum exhibit unique patterns of ELI. The dorsal striatopallidum contained far less ELI, whereas the archistriatum contained far more than would be expected on the basis of previous studies of opioid peptides in other avian species, including pigeons, chickens, and songbirds. These differences may reflect neural specializations unique to the budgerigar that contribute to the extraordinary flexibility of the vocal motor system of this species to acquire socially significant stimuli throughout life.

“Megabacteriosis” is a condition affecting many psittacine and nonpsittacine birds for which an effective, reliable therapy and means of prevention have not been developed. Megabacteriosis has been associated with a chronic wasting condition in the budgerigar (Melopsittacus undulatus) termed “going light,” but the organism also has been detected in clinically healthy, thriving birds. In this study, removing eggs from the nests of megabacteria-positive adult budgerigars and hand-raising hatchlings under isolation conditions prevented transmission of megabacteria in all offspring. Staining fecal smears and histologic tissues with Calcofluor White-M2R also was shown to be a reliable means of demonstrating megabacteria. Hand-raising budgerigar hatchlings, and those of other avian species in which megabacteriosis is considered to be of concern, is a potentially valuable method of producing offspring that are free of this organism.

The distribution of tyrosine hydroxylase (TH) was mapped out in cells and fibers of the budgerigar (Melopsittacus undulatus) brain. Special attention was given to vocal control and auditory nuclei because budgerigars are a psittacine species in which both males and females are capable of lifelong vocal learning (Farabaugh et al. [1994] J. Comp. Psychol 108:81-92). The results show that TH staining in the central nucleus of the anterior archistriatum (AAc) resembled that of surrounding archistriatal fields, except for portions of the ventral archistriatum, which exhibited substantially more TH+ fibers. Fewer fibers and fiber baskets are present in the central nucleus of the lateral neostriatum (NLc) than in surrounding fields. Both the oval nuclei of the ventral hyperstriatum (HVo) and anterior neostriatum (NAo) exhibit less fiber staining than surrounding fields whereas fiber staining in the medial NAo (NAom) and magnicellular nucleus of the parolfactory lobe (LPOm) resemble that of surrounding fields. Staining in primary telencephalic auditory nuclei was extremely low. The only sex difference observed was slightly increased TH staining in LPOm of females compared with surrounding fields on some tissue sections. These findings are in contrast to previous findings in zebra finch (Poephila guttata), a close ended vocal learning songbird in which TH staining in vocal nuclei increases during development and remains greater than surrounding fields throughout adulthood. The present results therefore support the view that catecholamines act to inhibit vocal plasticity in adult vocal learning species. Several unique features of TH-immunoreactive (ir) cell groups were observed in the brainstem including sparsely scattered TH-ir somata immediately adjacent to the third ventricle, within the tectum, basal forebrain, archistriatum, and caudal neostriatum, and in the hippocampus. These latter populations have not been described in other avian species and resemble features of the catecholamine system generally found in either reptiles or mammals.