15 22 January 2020

Joint Pathology Center Veterinary Pathology Services

WEDNESDAY SLIDE CONFERENCE 2019-2020

C o n f e r e n c e 15 22 January 2020

Dr. Cory Brayton, DVM, PhD, DACVP, DACLAM, Director, Phenotypic Core Associate Professor of Molecular and Comparative and Pathobiology Johns Hopkins School of Medicine Baltimore MD

CASE I: 16N131-1 (JPC 4128009). Microscopic Description: In sections of brain, there was a severe inflammatory Signalment: 3.5mo old, female, NOD.Cg- infiltrate composed exclusively of mature scid tm1Wjl Prkdc Il2rg /SzJ (NOD-SCID- and degenerate neutrophils within the third gamma/NSG) mouse (Mus musculus) and lateral ventricles, extending into the History: This mouse was xenografted in the subjacent neuropil of the hippocampus and mammary fat pad at 6 weeks of age with cerebrum with associated fragmentation and tumor cells from a breast cancer patient. The rarefaction of the neuropil. Several colonies mouse presented moribund, hunched and of short rod-shaped bacteria with peripheral scruffy two months later, and was clearing were observed within areas of subsequently euthanized. necrosis. The meninges were expanded with a mild inflammatory infiltrate composed Gross Pathology: The xenograft tumor was predominantly of neutrophils. In sections of not observed at gross necropsy. The mouse lung, multiple arteries contained variably was in poor body condition, with marked sized accumulations of fibrin, neutrophils, depletion of external and internal adipose and fewer macrophages, some containing stores. The lungs were mottled dark red, and similar rod-shaped bacteria. The the spleen was dark red to black and smaller perivascular interstitium and alveolar walls than normal. The small intestine contained were multifocally thickened with few small amounts of mucous, and few fecal neutrophils and macrophages, some of pellets were present in the descending colon. which contained intracytoplasmic bacteria. In sections of liver, there were few small Laboratory results: N/A. inflammatory foci composed of degenerate neutrophils associated with hepatocellular

3. Liver: Hepatitis, suppurative and embolic, multifocal, minimal, with rod-shaped bacterial colonies.

4. Bone marrow: Myeloid hyperplasia, diffuse, marked.

Contributor’s Comment: Klebsiella spp. are gram-negative rod-shaped bacteria that are typically commensals in mice, and are not a significant cause of naturally occurring disease. K. oxytoca is ubiquitous in the environment, and can be isolated from the gastrointestinal tract, nasopharynx, lung, Brain, NOD SCID mouse: Multiple sections of the brain are submitted with cerebellum and brainstem skin, and mucous membranes of healthy (top left), diencephalon (middle) and telencephalon animals and humans.2,7,14 However, these (inverted at bottom right). The lateral ventricles organisms may become opportunistic (black arrows), third ventricle (blue arrow) and fourth ventricle (green arrow) are distended and pathogens in certain situations, and in both contain a cellular exudate. (HE, 5X). humans and animals infections with K.

pneumoniae and oxytoca are most necrosis and rod-shaped bacteria, and there commonly associated with clinical disease. were increased numbers of inflammatory Klebsiella oxytoca can cause suppurative cell infiltrates within portal areas, composed lesions in various organ systems in mice, primarily of lymphocytes, macrophages, and particularly the reproductive tract, where it fewer neutrophils. Occasionally rod-shaped has been associated with suppurative bacteria were observed in Kupffer cells. In endometritis, salpingitis, and perioophortitis sections of bone marrow, there was diffuse often progressing to peritonitis and abscess and marked myeloid hyperplasia. Hucker- formation. Other reported opportunistic Twort gram stain on sections of brain infections in rats and mice include perianal revealed gram negative rod-shaped bacteria. dermatitis, otitis media, cystitis, pyelonephritis, keratoconjunctivitis, Contributor’s Morphologic Diagnosis: 1. Brain: Meningoencephalitis, suppurative, Harderian gland adenitis, oral infection, locally extensive, severe, with rod-shaped subcutaneous, abdominal and hepatic abscesses, pneumonia, meningitis, bacterial colonies. endotoxemia and septicemia. 1,3,8,14 In 2. Lung: Pneumonia, suppurative and humans, Klebsiella oxytoca and pneumoniae embolic, multifocal, moderate, with rod- are considered important causes of shaped bacterial colonies. nosocomial infections in hospitalized patients, and has been implicated in community acquired pneumonia, adult and

Klebsiella spp. infection. Suppurative otitis media, urogenital tract infections, and pneumonia have been reported in substrains of C3H/HeJ mice and NMRI- Foxn1nu mice, and LWE.1AR1 rats, and chronic renal inflammatory lesions and ascending urinary tract infections in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. 1,2,4,7,14 C3H/HeJ substrains are hyporesponsive to lipopolysaccharide (LPS) produced by gram-negative bacteria, due to a single Brain, NOD SCID mouse: Neutrophils occupy the amino acid substitution in the toll-like lateral ventricle and infiltrate the adjacent nu parenchyma, resulting in cavitation and thrombosis receptor 4 (TLR4) protein, NMRI- Foxn1 of parenchymal capillaries (arrows). (HE, 200X). (nude) mice lack a thymus and are therefore (Photo courtesy of: In Vivo Animal Core, Unit for T-cell deficient, and LEW.1AR-iddm rats Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109 are at risk for infection secondary to http://animalcare.umich.edu/business-services/vivo- development of diabetes mellitus. Finally, animal-core) NSG mice have multiple mutations neonatal sepsis and bacteremia, septic including the Prkdc severe combined arthritis, soft tissue abscesses, and urinary immune deficiency (scid) mutation, IL2 tract infections, and chronic nasal gamma deficiency, mutation of the C5 infections.1,2,10 K. oxytoca is also suspected complement gene, and a novel MHC to be the etiologic agent responsible for haplotype which leads to lack of normal T antibiotic-associated hemorrhagic colitis (AAHC) in humans, which occurs following antibiotic therapy and is characterized by bloody diarrhea, abdominal cramping and segmental hemorrhagic typhlocolitis 1,2,14. Antibiotic therapy has been shown to promote abnormal colonization by Klebsiella spp. in humans1 and rodents,5 and an animal model of AAHC has been developed using oral administration of antibiotics in rats followed by oral infection Brain, NOD SCID mouse: Neutrophils occupy the 6 with K. oxytoca. lateral ventricle and infiltrate the adjacent parenchyma, resulting in cavitation and thrombosis Immune status of the host plays an integral of parenchymal capillaries (arrows). (HE, 200X). role in the pathogenesis of disease by (Photo courtesy of: In Vivo Animal Core, Unit for Laboratory Animal Medicine, University of Michigan Klebsiella spp. Certain strains and Medical School, Ann Arbor, MI 48109 genetically modified mouse lines are more http://animalcare.umich.edu/business-services/vivo- prone to developing lesions associated with animal-core)

3 and B lymphocytes and NK cells, and staining, the observed peripheral clearing deficient cytokine and complement signaling around the gram-negative, rod-shaped and function.4 Increasing age may also play bacterial organisms is characteristic of a contributory role in pathogenesis of Klebsiella spp. The affected animal in this Klebsiella infection, as suppurative case was a NSG mouse, which as previously reproductive lesions due to K. oxytoca discussed is a severely immunodeficient infection have been reported in National mouse model prone to opportunistic Toxicology Program (NTP) chronic infection. These mice are commonly used to chemical carcinogenesis bioassays.3 model development, progression, and metastasis of human tumor cells in vivo, Klebsiella exerts its pathogenic effects by through xenotransplantation experiments. taking advantage of an immunosuppressed or immunocompromised host, and through the use of several virulence factors. The presence of a distinct polysaccharide capsule observed as a peripheral clearing on routine light microscopy is correlated with virulence of the organism, enabling it to resist phagocytosis and bactericidal components of serum.1,7,10 In addition, several in vitro studies in humans and in mice have shown that K. oxytoca produces a cytotoxin, tilivalline, which induces cell death through inhibition of DNA synthesis.2,14 Lung, NOD SCID mouse: Septic fibrinocellular thrombi are present within the lumen of pulmonary Furthermore, genomic studies on tilivalline- arterioles. Incorporated macrophages and producing K. oxytoca in both humans and neutrophils often contain encapsulated bacilli within mice revealed a number of genes associated their cytoplasm. (HE, 400X). (Photo courtesy of: In Vivo Animal Core, Unit for Laboratory Animal 2 with virulence potential. Finally, a substrain Medicine, University of Michigan Medical School, of K. oxytoca (TNM3) has been shown to Ann Arbor, MI 48109 produce an immunosuppressive http://animalcare.umich.edu/business-services/vivo- polysaccharide, AZ9, which is associated The source of the infection is unknown, but with decreased IL4 and IFNg responses it is possible that K. oxytoca was introduced leading to an overall depressed Th2-type through human contact as this organism can 1 immune response.11,12 be spread from humans to rodents, or through environmental contamination. One In the present case, lesions were clearly major concern with the presence of such representative of a septicemic process with opportunistic organisms in animals multiple suppurative and embolic lesions in maintained with a well-defined hygienic several organs including the lung, liver, and status (specific pathogen-free) is that they brain, and a myeloid response in the bone are becoming more common and relevant as marrow. By light microscopy and gram causes of disease in such colonies,1 so

knowledge of these organisms is imperative JPC Diagnosis: Brain: Ventriculitis, to maintaining high standards of animal periventriculitis, and meningitis, necrotizing welfare and research quality. Furthermore, and suppurative, multifocal to coalescing, in experiments utilizing severe, with vasculitis, thrombosis gliosis, immunocompromised mice for serial tumor and numerous bacilli. passage or xenograftment, transfer of tumor

cells or other biologic material contaminated JPC Comment: The contributor has with infectious agents such as Klebsiella is a provided an outstanding and thorough potential significant risk factor to the health review of this opportunistic infection in recipient mice, as well as the quality of the laboratory rodents, especially experimental outcome due to morbidity and immunosuppressed models. mortality secondary to opportunistic, and Tilvallin, as mentioned bythe contributor, is unexpected, infection. a gene product of K. oxytoca which induces apoptosis and loss of barrier integrity in Contributing Institution: vitro in human epithelial cells, which In Vivo Animal Core, Unit for Laboratory suggests a possible pathogenesis inhuman Animal Medicine antibiotic-associated hemorrhagic colitis (AAHC).12 Further investigation of the University of Michigan Medical School biosynthesis of this compound has Ann Arbor, MI 48109 demonstrated a number of other secondary metabolites, also pyrrolobenzodiazpines http://animalcare.umich.edu/business- (PBD), including tilmysin and culdesacin. services/vivo-animal-core The combination of tilmysin with indole actually yields tilvallin. The PBD family of compounds are potent cytotoxic agents which demonstrate both antibacterial and anticancer activity due to DNA alkylation Lung, NOD SCID mouse: Septic fibrinocellular and formation of PBD-DNA adducts. In thrombi are present within the lumen of pulmonary arterioles. Incorporated macrophages and addition, tilvallin is also a microtubule neutrophils often contain encapsulated bacilli within stabilizing agent, a class of compunds that their cytoplasm. (HE, 400X). (Photo courtesy of: In shift the balance of cellular tubulin from Vivo Animal Core, Unit for Laboratory Animal Medicine, University of Michigan Medical School, soluble to polymerized and are widely used Ann Arbor, MI 48109 as anticancer agents. This class also http://animalcare.umich.edu/business-services/vivo- includes taxol, a widely used

Cerebrum, NOD SCID mouse. Numerous discrete chemotherapeutic12. encapsulated gram-negative bacilli are present within the ventricular exudate. (Hucker-Twort, 400X) (Photo courtesy of: In Vivo Animal Core, Unit for In 2011, the Enterobacteriaceae genus Laboratory Animal Medicine, University of Michigan Raoultella (named after the French Medical School, Ann Arbor, MI 48109 bacteriologist Didier Raoult) was separated http://animalcare.umich.edu/business-services/vivo- animal-core) from Klebsiella through the use of molecular techniques, as well as the identification of

growth at 10o C and use of L-sorbose as a References carbon source). This genus include four species: Raoultella ornithinolytica, R. 1 Bleich A, Kirsch P, Sahly H, Fahey J, 9 Smoczek A, Hedrich HJ, et al.: planticola, R. terrigena, and R. electrica. Klebsiella oxytoca: opportunistic These bacilli are found in plants and soli in infections in laboratory rodents. Lab aquatic environments. R. ornithinolytica Anim 2008:42(3):369-375. and R. planticola are considered emerging 2 Darby A, Lertpiriyapong K, Sarkar U, human pathogen, which results in biliary Seneviratne U, Park DS, Gamazon tract infections in elderly or ER, et al.: Cytotoxic and pathogenic immunosuppressed patients with properties of Klebsiella oxytoca malignancies or who have undergone isolated from laboratory animals. invasive procedures. It is considered likely PLoS One 2014:9(7):e100542. that a number of Klebsiella infections 3 Davis JK, Gaertner DJ, Cox NR, Lindsey diagnosed historically may actually be of JR, Cassell GH, Davidson MK, et 9 bacilli of this genus. al.: The role of Klebsiella oxytoca in utero-ovarian infection of B6C3F1 The moderator discussed Rodentibacter mice. Lab Anim Sci 1987:37(2):159- pneumotropica as a common opportunist in 166. immunosuppressed mice (which is usually difficult to see on HE and special stains), 4 Foreman O, Kavirayani AM, Griffey SM, Reader R, Shultz LD: Opportunistic which highlights that the name of this bacterial infections in breeding common opportunist has changed within the colonies of the NSG mouse strain. last few years (for those who are trying to Vet Pathol 2011:48(2):495-499. keep up with the microbiologists. The moderator also commented on the 5 Hansen AK: Antibiotic treatment of nude rats and its impact on the aerobic description of a “smaller than normal” bacterial flora. Lab Anim spleen, as the normal weight of the spleen of 1995:29(1):37-44. an immunocompent mouse is 0.2g, and of a NOD-SCID mouse is 0.02g, highlighting not 6 Hogenauer C, Langner C, Beubler E, Lippe IT, Schicho R, Gorkiewicz G, only the size difference of spleens, but the et al.: Klebsiella oxytoca as a need to use absolute weights in this species. causative organism of antibiotic- As this was a xenograft animal, the associated hemorrhagic colitis. N moderator wondered about the possibility of Engl J Med 2006:355(23):2418- irradiation prior to engraftment in this 2426. individual further complicating this picture. 7 MacArthur CJ, Pillers DA, Pang J, Unfortunately lab data was not available in Degagne JM, Kempton JB, Trune this case to distinguish between K. oxtyoca DR: Gram-negative pathogen and K. pneumoniae in this case, so the Klebsiella oxytoca is associated with moderator was loathe to pick one species spontaneous chronic otitis media in over the other. Toll-like receptor 4-deficient

C3H/HeJ mice. Acta Otolaryngol 14 Whary M, Baumgarth, N., Fox, J. G., 2008:128(2):132-138. Barthold, S. W.: Biology and Diseases of Mice. In: Fox JF, 8 Percy DH, Barthold S. W.: Pathology of Anterson, L. C., Otto, G. M., Laboratory Rodents and Rabbits. 4th Pritchett-Corning, K. R., Whary, M. ed. Ames, IA: Blackwell Publishing; T., ed. Laboratory Animal Medicine. 2016: 50-72. 3rd ed. Amsterdam: Academic Press; 2015. 9. Ponce-Alonse M. Rodrigues-Rojas L, del Campo R, Canton, R, Morosini MI. CASE II: MS18-3968 (JPC 4136806). Comparison of different methods for identificatio of species of the Signalment: Adult, male, albino, genus Raoultella: report of 11 cases CYBB[ko] mouse, Mus musculus of Rasoultella causing bacteraemia and literature review. Clin History: NSG.Cybb[KO] mouse observed Microbiol Infect 2016; 22:252-257. with scruffy hair coat, slightly hunched 10 Sahly H, Podschun R: Clinical, posture and pale ears. Mice of this strain bacteriological, and serological have been spontaneously dying. These mice aspects of Klebsiella infections and are on corn cob bedding and are provided their spondylarthropathic sequelae. with TMS antibiotic water. The feed is Clin Diagn Lab Immunol regular rodent chow and is autoclaved with 1997:4(4):393-399. the cage setup. The antibiotic water is made 11 Sugihara R, Matsumoto Y, Ohmori H: by sterilizing tap water in the autoclave and Suppression of IgE antibody then adding TMS at the room in a Biological response in mice by a Safety Cabinet (BSC). The cages are sterile polysaccharide, AZ9, produced by when they arrive at the room with aseptic Klebsiella oxytoca strain TNM3. technique used to transfer the mice from the Immunopharmacol Immunotoxicol 2002:24(2):245-254. dirty cage to the clean cage. All manipulation is performed in a BSC. 12 Sugihara R, Oiso Y, Matsumoto Y, Ohmori H: Production of an immunosuppressive polysaccharide, AZ9, in the culture of Klebsiella oxytoca strain TNM3. J Biosci Bioeng 2001:92(5):485-487. 13. Unterhauser K, Potil L, Scheiditz GS, KKeienesberger S. et al. Klebsiella oxytoca enterotoxins tilimycin and tilivalline have distinct host DNA- damaging and microtubule- Spleen and pancreas, CYBB[ko] mouse. A section of stabilizing activities. Proc Natl Acad spleen, pancreas, and mesentery is submitted. The Sci USA 2019; 116(9): 3774-3783. spleen is 4-5 times normal thickness and normal follicular and sinusoidal architecture is not evident. (HE, 5X)

Spleen, CYBB[ko] mouse. Effacing approximately 50% of the splenic architecture in a nodular pattern are large numbers of macrophages (often spindling), admixed with large numbers of neutrophils. Mitotic figures are common. (HE, 400X)

Gross Pathology: Presented for necropsy pale tan slightly raised masses scattered was a live adult male white mouse. The throughout. animal appeared to be in good nutritional Upon opening the pleural cavity, the lungs condition. The animal had a roughened hair contained numerous light red pinpoint coat, yet was alert and active in the transport scattered foci. There was a 1 mm in box. The animal was euthanized with CO2. diameter clear cyst in the right caudal lung Upon opening the carcass, adequate adipose lobe. stores were observed. The subcutis was Lesions were not observed in the brain, slightly tacky indicating mild dehydration. heart, kidneys, pancreas, the entire male Upon opening the peritoneal cavity, the reproductive tract, and the entire spleen was enlarged approximately three gastrointestinal tract that has scant times normal size and contained multifocal ingesta/digesta throughout and multiple to coalescing pale tan masses that expanded formed feces in the descending colon. above the capsular surface. The liver was also enlarged approximately three times Laboratory results: Microbiology yielded normal size with multifocal to coalescing Candida parapsilosis from the liver.

Microscopic Description: Spleen – Contributor’s Morphologic Diagnosis: Multifocal to coalescing granulomas with epitheliod macrophages, many with Spleen, splenitis, pyogranulomatous and neutrophils, and a coarse fibrous connective fibrosing, multifocal to coalescing, severe, tissue network are observed. In a number of chronic with intralesional yeast, Candida these are intracellular, one to a few, 1-5 um parapsilosis round to oval yeast organisms with a clear halo, and which a number are seen budding Contributor’s Comment: on PAS stain. The adjacent parenchyma has NSG.Cybb{KO} mice are a genetically a moderate granulocytic hyperplasia. Similar altered mouse strain that is severely lesions were observed in the liver, and yeast immunocompromised. NSG (NOD.Cg- were observed in the liver, kidneys Prkdscid Il2rgtm1Wjl/SzJ) is a lymphocyte (tubulitis) and lungs (alveolitis). deficient strain, and Cybb KO (CYBB gene is located on X-chromosome, and encodes

the gp91phox protein) is the most common

Spleen, CYBB[ko] mouse. The splenic red pulp is filled with immature granulocytes with a predominance of band cells (with doughnut-shaped nuclei), and fewer islands of hyperchromatic erythrocyte precursors as well as megakaryocytes. (HE, 400X)

form of Chronic Granulomatous Disease absent oxidase activity predisposes to a (CGD).12 CGD is an immunodeficiency with dysregulated immune response in a a defect in phagocytes (macrophages, dominant form.12 neutrophils) to produce reactive oxygen species (ROS) which are needed for CGD appears to be a dysregulated microbicidal activity. Normally, ROS are granulomatous inflammation in various generated by phagocyte NADPH oxidase. organs including the GI tract, respiratory This enzyme is composed of 5 subunits; 2 tract, as well as the eye and urinary tract. In plasma membranes and 3 cytosolic. some patients a true autoimmune disease Membrane bound are transmembrane was exhibited; systemic lupus glycoproteins (gp), one with 91kD mass erythematosus, rheumatoid arthritis, called gp91phox (phox for phagocyte oxidase; inflammatory bowel disease, Sjogren’s also known as NOX2[neutrophil oxidase-2]) syndrome and atopic dermatitis. and a 22kD gp called p22phox. These two Interestingly, a large percentage of human form a heterodimer. The 3 cytosolic patients did not show any evidence of 13 subunits (p40phox, p47phox, p67phox) form a infection. heterotrimer. The genes of the five Some patients with CGD suffer from a components are CYBB [cytochrome b beta] variety of recurrent bacterial and fungal located on the X-chromosome encoding diseases. The most common bacterial phox gp91 , CYBA [cytochrome b infections include Staphylococcus aureus, phox alpha]encoding p22 , NCF1[neutrophil Klebsiella spp., Burkholderia cepacia, phox cytosol factor] encoding p47 , NCF2 Serratia marcescens and Salmonella spp.9,14 phox encoding p67 , and NCF4 encoding Fungal infections include Aspergillus phox 10 p40 . fumigatus, A. nidulans, A. niger, A. flavus, The most common form of CGD involves Zygomycota (primarily Rhizopus spp.), the CYBB gene (deletion, frameshift, Candida spp., Trichosporon spp., nonsense, missense, splice site mutation) Paecilomyces spp., Scedosporium spp., that affects mostly males because of the Penicillium spp., Acremonium spp., predominant mode of genetic transmission Alternaria spp., Inonotus spp., Exophiala, (X- chromosome).8 Interestingly, Chrysosporium spp., Fusarium spp., 7,15 heterozygous mothers of affected males will Microascus spp., and Hansela spp. A. carry a proportion of innate immune cells nidulans seems to have a unique interaction 7 that are fully oxidase deficient. These with CGD hosts. Geosmithia argillacea has individuals are prone to both infectious recently been shown to be mistakenly 6 complications and autoimmune diseases. Of identified as Paecilomyces spp. in the past. note, the risk of developing autoimmune Dimorphic yeast form infections are disease was not at all related to degree of exceedingly rare with only one reported case 14 residual oxidase activity as it is in each of Coccidioides immitis, Histoplasma 14 14 developing an infection. This suggests that capsulatum, and Sporothrix schenckii. even the presence of a minority of cells with

In this case Candida parapsilosis was the sensu stricto, C. orthopsilosis and C. pathogenic yeast identified. Candida species metapsilosis.2,10 Polymorphisms in the genes are mainly found in the gastrointestinal tract COX3 (mitochondrial gene cytochrome of humans9. C. parapsilosis is found oxidase subunit 3), SADH (secondary frequently on the skin and hands11. Although alcohol dehydrogenase) and SYA1 (putative commensal, they can become pathogenic alanyl-tRNA synthetase) distinguish the when host defense mechanisms or three species.5 Virulence of C. parapsilosis anatomical barriers are compromised.10 C. is related to factors such as cell wall parapsilosis is currently the second leading constituents, adhesion to biotic and abiotic cause of candidemia which is associated structures leading to biofilm formation, and with a high morbidity and mortality rate in extracellular enzymes such as aspartic humans.12 In CGD humans, Candida species proteases, phospholipases and lipases. were isolated from meningitis, fungemia, Aspartic proteases promote tissue lymphadenitis.13 colonization and invasion by rupturing host mucosal membranes. They may also aid in C. parapsilosis is actually a complex of 3 dispersion of biofilms. Phospholipases distinct genetic species: C. parapsilosis promote rupture of host cell membranes. Lipases help with acquiring nutrition, support fungal growth, mediate adhesion to cells and tissues, and coordinate yeast interactions with enzymes and immune cells during the infectious process.11

Initial attachment of Candida to host cells is followed by cell division, proliferation (forming yeast and pseudohyphae) and subsequent biofilm formation. Biofilm formation is an important virulence Spleen, CYBB[ko] mouse. Macrophages contain one or more 3-5um intracytoplasmic factor as it confers yeasts. (HE, 400X) significant resistance to antifungal therapy by

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JPC Diagnosis: 1. Spleen: Splenitis, pyogranulomatous, diffuse, severe, with intrahistiocytic yeasts. Spleen, CYBB[ko] mouse. A silver stain demonstrates budding yeasts. (GMS, 400X) 2. Spleen, red pulp: Extramedullary limiting the penetration of substances hematopoiesis, diffuse, severe. through the matrix (water, ions, JPC Comment: The contributor has carbohydrates, proteins and nucleic acids) provided an excellent review of this animal and protecting fungal cells from host model of an uncommon inherited primary 14 immune responses . Morphologically, C. immunodeficiency, the mechanism of the parapsilosis is unable to form true hyphae, immunodeficiency, as well as a review of but forming pseudohyphae is associated Candida sp. in general as well as C. 11 with virulence. parasilopsis, the particular pathogen in this This case is somewhat baffling given that case. the animal was in a sterile environment with sterile bedding, caging and water yet still As mentioned by the contributor, chronic was exposed to Candida. However, in granulomatous disease (CGD) is an humans, C. parapsilosis is frequently uncommon X-linked immunodeficiency in isolated from hands and from sterile body humans resulting in frequent bacterial and sites. Thus, human transmission may have fiungal infections. Patients with CGD may been possible. Overall, three mice developed possess a defect in one of four different 8,10 similar lesions, with two having structural proteins in NADPH oxidase. recognizable yeast organisms on NADPH oxidase catalyzes the transfer of a histopathology. One should be vigilant and single electron from NADPH to molecular be alert for uncommon infections in a oxygen, generating one of four reactive laboratory setting. oxygen species, from the superoxide radical, including the peroxynitrite anion, hydroxyl Contributing Institution: anion, hypochlorous acid, and nitryl chloride. 8,10

The contributor mentions the many types of fungal infection which are seen in References: immunsuppressed individuals. The reason for this is that mononuclear phagocyte 1. Antachopoulos C. Invasive fungal activity is the major driver of resistance to infections in congenital systemic mycoses. Patients suffering from immunodeficiencies. Clin Microbiol systemic mycosis have shown consistent Infect. 2010; 16:1335-1342. benefit from concurrent administration of 2. Bertini A, De Bernardis F, Hensgens macrophage colony-stimulating factor. LAM, Sandini S, Senesi S, Tavanti A. Patients with CGD are treated with Comparison of Candida parapsilosis, continuous antibacterial and antifungal Candida orthopsilosis, and Candida medications, and the only current long-term metapsilosis adhesive properties and treatment is allogeneic hematopoitic stem pathogenicity. Int J Med Microbiol. cell transplants. 2013; 303:98-103. 3. Bonassoli LA, Bertoli M, Scidzinski Interestingly, this WSC conference TIE. High frequency of Candida submission is not the JPC’s first encounter parapsilosis on the hands of healthy with this mutant strain of mouse from the hosts. J Hosp Infect. 2005; 59:159- National Institutes of Health, Following a 162. rotation at the Dept. of Veterinary Resources 4. Cavalheiro M, Cacho Teixeira M. at the NIH, then resident Dr. Shannon Lacy Candida biofilms: Threats, (himself a former resident coodinator of the challenges, and promising strategies. Wednesday Slide Conference published an Frontiers Med 2018; 5:1-15. article about infection seen in this same 5. De Aguiar Cordeiro R, Alencar S, de strain of knockout mice with another Souza Collares Maia Castelo-Branco, saprophytic fungus, Trichosporoon beigelli.8 et al. Candida parapsilosis complex Like Candida, Trichosporoon is part of the in veterinary medicine: A historical normal flora of human skin and GI tract. overview, biology, virulence This was the first report of disseminated attributes and antifungal susceptibility trichosporoonosis in the laboratory mice of traits. Vet Microbiol. 2017; 212:22- any immunosuppressed strain.8 30. 6. DeRavin SS, Challipalli M, Anderson The moderator noted that in this case, the V, Shea YR, Marciano B, Hilligross yeasts in the submitted slide did not make D, Marquesen M, DeCastro R, et al. either pseudohyphae or hyphae, which is Geosmithia argillacea: An emerging consistent with the literature. C. cause of invasive mycosis in human parapsilosis apparently makes chronic granulomatous disease. Clin pseudohyphae in culture and within biofilms Infect Dis. 2011; 52(6):e136-e143. and not in tissue. It also does not produce 7. Henriet S, Verweij PE, Holland SM, true hyphae. Warris A. Invasive fungal infections in patients with chronic

granulomatous disease. In: Curtis N, 14. Trotter JR, Sriaroon P, Berman D, et al. eds. Advances in Experimental Petrovic A, Leiding JW. Sporothrix Medicine and Biology. Hot topics in schenckii lymphadenitis in a male infection and immunity in children IX. with X-linked chronic granulomatous New York, NY; Springer disease. J Clin Immunol. 2014; 34:49- Science;2013: 27-55. 52. 8. Lacy SH, Garnder DJ, Olson LC, 15. Winkelstein JA, Marino MC, Ding L, Holland SM, Bryant MA. Johnston RB, Boyle J, Curnette J, Disseminated trichosporonosis in a Gallin JI, Malech HL, et al. Chronic murine model of chronic granulomatous disease. Report on a granulomatous disase. Comp Med national registry of 268 patients. 2003; 53(3): 303-308. Medicine (Baltimore). 2000; 9. Rider NL, Jameson MB, Creech CB. 79(3):155-169. Chronic granulomatous disease:

Epidemiology, pathophysiology, and genetic basis of disease. JPIDS. 2018; CASE III: 17-1438 1-2 (JPC 4101223) 7(S1):S2-S5. 10. Roos D. Chronic granulomatous Signalment: Two-month-old, intact female disease. Br Med Bull. 2016; 118:53- mouse, Mus musculus. 66. History: The mouse was purchased from a 11. Silva S, Negri M, Henriques M, non-conventional vendor (pet shop) and it Oliveira R, Williams DW, Azeredo J. was enrolled in a study as model of Candida glabrata, Candida autoimmune colitis. The animal was parapsilosis, and Candida submitted for euthanasia and necropsy given tropicalis:biology, epidemiology, the loss of body weight (10% in the previous pathogenicity and antifungal 2 weeks) and hunched posture. No other resistance. FEMS Microbiol Rev mouse in the group, from the same source 2012; 36:288-305. and in the same experimental conditions, 12. Sweeney CL, Choi U, Liu C, Koontz exhibited any similar or different sign of S, Ha S_K, Malech HL. CRISPR- disease. mediated knockout of Cybb in NSG mice establishes a model of chronic Gross Pathology: The lung lobes are granulomatous disease for human diffusely expanded, firm and mottled stem-cell gene therapy transplants. (Fig.1). Within the cranial regions there are Hum Gene Therap. 2017; 28(7):565- 575. 13. Thomas DC. How the phagocyte NADPH oxidase regulates innate immunity. Free Rad Bio Med. 2018; 125:44-52.

lined by one or more layer of cuboidal to columnar epithelium (bronchiolitis obliterans). Multifocally the respiratory epithelium is replaced by one or more layers of cells with features of squamous epithelium (squamous metaplasia, Fig. 3). Large cuffs composed of lymphocytes and plasmacells surround the intrapulmonary branches of the bronchial tree. Protein rich fluid is multifocally present within few Lungs, mouse. There are extensive areas which is alveoli (alveolar edema). most prominent in the left lung. Airways are multifocally markedly ectatic, nodular in appearance and filled with exudate. (Photo courtesy of: Contributor’s Morphologic Diagnosis: Laboratory of Comparative Pathology; Hospital for Special Surgery, Memorial Sloan Kettering Cancer Lungs: suppurative bronchopneumonia, Center, The Rockefeller University, Weill Cornell subacute, severe with bronchiectasis, Medicine. https://www.mskcc.org/research- bronchiolectasis, bronchiolitis obliterans and areas/programs-centers/comparative-medicine- squamous metaplasia, consistent with M. pathology) pulmonis infection multiple grey-white nodules measuring about 0.5 to 1 mm in diameter (consistent with bronchiectasis). Contributor’s Comment: Mycoplasma is a genus of bacteria belonging to the order Laboratory results: PCR for Mycoplasma Mycoplasmatales, family spp. on fresh frozen lung tissue was positive Mycoplasmataceae, class Mollicutes. The class name indicates the lack of a wall Microscopic Description: About 80 % of around the cell membrane, which is a the parenchyma is affected in a multifocal to peculiar feature of these bacteria. As such coalescing fashion by the infiltration and accumulation of a large number of mostly viable and occasionally degenerated neutrophils, admixed with few foamy macrophages, obliterating the lumen of alveoli, bronchioles and bronchi (Fig.2). The adjacent parenchyma is atelectatic. Few bronchial and bronchiolar structures exhibit distorted outlines with dilation (bronchiectasis and bronchiolectasis), thickening of the lining epithelium with piling up of nuclei (hyperplasia), and Lungs, mouse. Multiple sections of lung are submitted. Inflammatory changes are focused on luminal narrowing by polypoid-like airways and affect from 80-100% of each section. structures, formed by a fibrous stalk and (HE, 6X)

Lungs, mouse. Multiple sections of lung are submitted. Higher magnification of an affected lobe with a markedly dilated, exudate-filled airway (bronchiectasis). (HE, 15X)

they are classified as gram-indeterminate. disease in subclinically infected mice. The Mycoplasmae are divided into 2 clusters: modern husbandry standards of mouse hemotropic and pneumoniae, based on 16S facilities and health monitoring plans in sequencing. research institutions contributed to decrease the incidence of mycoplasmosis despite the The “pneumoniae” Mycoplasma are often rather high percentage of infected animals. non-pathogenic and are found in the genital and respiratory tracts. Mycoplasma species Overall, mice are less susceptible than that are hosted in laboratory mice are: M. laboratory rats to the disease, however pulmonis, M. arthritidis, M. neurolyticum, susceptibility to the disease varies upon the M. collis, M. muris, however M. pulmonis mouse strain, with the B6 being resistant and only is a significant pathogen. the C3H quite sensitive. Moreover, females seem to develop more severe forms. The Factors such as Sendai virus infection, high natural disease in immunocompetent mice is concentration of ammonia in the usually characterized by weight loss, environment and co-infection with respiratory symptoms and head tilt or Pasteurella pneumotropica play an vestibular signs in case of concomitant important role in the clinical onset of the

otitis. Immunodeficient mice on the other Variably sized pearl-white to tan nodules hand develop arthritis.8 can be observed. On cut section, they reveal a wall and a lumen partially or completely Colonization of the respiratory tract is obliterated by exudate that correspond to mediated by adhesion through special dilated bronchial structures (bronchiectasis). organelles of Mycoplasma organism to cilia, with consequent ciliostasis and retention of Histologically, the main and most consistent mucus.7 features are the luminal collection of viable and degenerated neutrophils in the nasal Grossly, mucopurulent exudate can be seen cavities, tympanic bullae, lower airways and in the tympanic bulla, nasal passages and the infiltration of lymphocytes and plasma airways. When the disease is subacute or cells forming cuffs around bronchi and even chronic consolidation of the lung bronchioles. Abscess formation as well as parenchyma and mottling, especially in the squamous metaplasia of the respiratory cranioventral regions can be appreciated. epithelium can be observed in chronic cases.

Lungs, mouse. The ectatic lumen (top) is filled with numerous viable and degenerate neutrophils. The hyperplastic epithelium (center) is infiltrated with neutrophils and lymphocytes, and below the wall of smooth muscle, there is accumulation of large numbers of lymphocytes and plasma cells. (HE, 242X)

The lymphoplasmacytic infiltrate is reported https://www.mskcc.org/research- to be more prominent in rats than in mice.8 areas/programs-centers/comparative- The mycoplasmal membrane is decorated medicine-pathology with high frequency variation antigens which act as superantigens, stimulating the humoral response in the infected host. JPC Diagnosis: Spleen: Splenitis, Lymphoid hyperplasia around the airways is pyogranulomatous, diffuse, severe, with a consistent feature of the enzootic intrahistiocytic yeasts. pneumonia of swine, caused by M. hyopneumoniae.7 2. Spleen, red pulp: Granulocytic and myeloid hyperplasia, diffuse, severe. Mycoplasma have the ability to establish infection thanks to several bacterial factors 3. Spleen, white pulp: Lymphoid that prevent phagocytosis and killing by hypoplasia, diffuse severe (consistent with macrophages, which are the first line genotype). defense, and increased adherence to host’s mucosal surfaces.3,6,9 Capsular 4 polysaccharide is an antiphagocytic factor. JPC Comment: Mycoplasma, in all their Mycoplasma pulmonis produces a minimalized finery, embody much of what polysaccharide called EPS-I which has roles we know about what is actually necessary in cytoadherence, protection from for the development of cellular life. In complement, inhibition of biofilm 1962, the National Aeronautic Space 1,2 formation. It acts moreover as second Administration (NASA) embarked on a shield defense against macrophage binding continuing search for extraterrestial life, and and phagocytosis. EPS-I appears to provide suggesting that if found, it would be maximal defense against host response when extremely simple, leading a generation of 8 present together with a long Vsa protein. scholars to look on our own planet for what Vsa are a family of surface lipoproteins that form of life would resemble.9 In doing 10,11-13 produced by mycoplasmas. They vary so, the early investigations into in phase and size. Size variation in particular mycoplasma, the simplest form of life with reduces binding of the bacteria by the ability of independent growth in artificial macrophages beside inhibiting biofilm media, were purued. formation and protecting from complement action similarly to EPS-I. Mycoplasa are indeed the essence of a “stripped down” life form, considered a Contributing Institution: “minimal” cell. They did not start as simple organisms at the base of the evolutionary Laboratory of Comparative Pathology; tree which did not progress over billions of Hospital for Special Surgery, Memorial year, but evolved contrary to typical Sloan Kettering Cancer Center, The evolution, shedding large parts of its Rockefeller University, Weill Cornell genome, as well as an independent lifestyle Medicine.

for a very specialized parasitic one, nutrients (and in the proper concentrations) becoming ever simpler and representing the from the host to survive, a feautre that absolute minimal requirements for cellular prevented their growth in the laboratory for life. many years. Many continue to be fastidious in their requirements, often failing to grow Three species of mycoplasma have the as a result of mild “overdosing” of required smallest genome of all cellular life forms, amino acids or other nutrients in media.9 with M. genitalium, a cause of urethritis in (They are however, excellent parasites of humans, having the smallest genome at cell cultures, with estimates of up to as 580,076 base pairs (contained in circular many of 80% of cell cultures globally DNA).5,9 In order to achieve this amazing containing living mycoplasmas as “part of housecleaning, mycoplasmas have sacrificed doing business”.)9 many of their genes, relegating themselves to a very particular parasitic (or in the case Additional gene deletions have affected of some insect mycoplasmas) symbiont energy metabolism, leaving mycoplasmas to lifestyles. depend on glycolysis as their main, however ineffective means of energy production. One of the obvious results of the shedding of They also are lacking in genes coding for “excess genes” is their lack of a cell wall. elements of the Kreb’s cycle or any Unlike most bacteria possess a cell wall, cell cytochromes, damning these processes as membrane, and cytoplasmic membrane, the “nice to have” but not essential for cellular 9 evolutionary transition from their life. presumptive gram-positive roots, mycoplasma have jettisoned both the cell One other way that mycoplasmas have wall and cytoplasmic membrane, making adopted a stripped down life cycle is their themselves osmotically fragile and unable to lack of “redundant” genes. For most cellular live in the extra-organism (or extracellular functions in traditional bacteria, such as environment for those species who live E.coli, each function as built-in redundancy, within other cells), but facilitating other with anywhere from 2-6 separate genes for interesting behavior such as direct fusion of the same function. Mycoplasmas in their membranes with their host ro target generally possess a single gene for each cells to facilitate homeostatic or cytopathic function, which when knocked out as part of endeavors).9 (The lack of the a cell wall is genomic investigation, most often results in also the reason why mycoplasma are death of the cell, or rarely, revision to non- 9 resistant to many traditional antibiotics, pathogenicity. which attack bacterial cell walls.) With all of this loss of genetic material, it is Another reduction cementing their parasitic interesting that M. genitalium, the cellular lifestyle is the deletion of all genes involved organism with the smallest genome in the in amino acid, fatty acid and cofactor world, yet possesses twice the number of biosynthesis. Mycoplasma must receive all genes that investigations in the “minimal

19 cell set” described by molecular biologists 4. Finlay BB, Falkow S. Common as the requirements for life and themes in microbial pathogenicity. reproduction.5 It would appear that Microbiol. Rev. 1989;53:210–230 Mycoplasma as a genus still has some 5. Glass JI, Merryman C, Wise KS, housecleaning to do. Hutchison III CA, Sith HO. Minimal cells, real and imagined. Cold One of the most interesting morphologic Spring Harbor Perspect Biol 2017; changes in this particular slide is the marked 9:a023861. hyperplasia of bronchiolar epithelium which 6. Hickman-Davis J, Michalek SM, appears to extend into adjacent alveoli by Gibbs-Erwin J, Lindsey JR. lepidic growth. While the cause of this Depletion of alveolar macrophages change is not evident, the moderator and exacerbates respiratory other renowned mouse pathologists with mycoplasmosis in mycoplasma- whom she consulted on this case suggested resistant C57BL mice but not the possibility of a concurrent virus which mycoplasma-sensitive C3H mice. may may not have been tested for, such as Infect. Immun. 1997;65:2278–2282 Sendai or pneumonia virus of mice. 7. Maxie G. Jubb Kennedy Palmer’s th Pathology of Domestic Animals 6 ed. Elsevier; 2015 vol. 2: 533-35. References: 8. Percy DH, Barthold SW. Mouse. In: Percy DH, Barthold SW , eds. 1. Bolland JR, Dybvig K. Mycoplasma Pathology of Laboratory Rodents pulmonis Vsa proteins and and Rabbits. 3rd ed. Ames, IA: polysaccharide modulate adherence Blackwell; 2007: 3–124. to pulmonary epithelial cells. FEMS 9. Razin S, Yogev D, Naot Y. Microbiol. Lett. 2012;331:25–30 Molecular Biology and 2. Bolland JR, Simmons WL, Pathogenicity of Mycoplasmas. Daubenspeck JM, Dybvig K. Micorbiol Mo Biol Rev 1998; 1094- Mycoplasma polysaccharide protects 1156. against complement. Microbiology. 10. Shaw BM, Daubenspeck JM, 2012;158:1867–1873 Simmons WL, Dybvig K. EPS-I 3. Davis JK, Davidson MK, Schoeb polysaccharide protects Mycoplasma TR, Lindsey JR. Decreased pulmonis from phagocytosis. FEMS intrapulmonary killing of Microbiol Lett. 2013 Jan;338(2):155- Mycoplasma pulmonis after short- 60. doi: 10.1111/1574-6968.12048. term exposure to NO2 is associated 11. Shaw BM, Simmons WL, Dybvig K. with damaged alveolar macrophages. The Vsa shield of Mycoplasma Amer. Rev. Resp. Dis. pulmonis is antiphagocytic. Infect. 1992;145:406–411 Immun. 2012;80:704–709

12. Simmons WL, Dybvig K. The Vsa from lytic effects of complement and proteins modulate susceptibility of gramicidin. Infect. Immun. Mycoplasma pulmonis to 2007;75:3696–3699. complement killing, hemadsorption, and adherence to polystyrene. Infect. Immun. 2003;71:5733–5738. CASE IV: 17N076 (JPC 4103914). 13. Simmons WL, Denison AM, Dybvig Signalment: ~6 months; Female; NOD.Cg- K. Resistance of Mycoplasma Prkdcscid IL2rgtm1Wjl/SzJ (aka NOD-scid- pulmonis to complement lysis is gamma, NOD-scid IL-2Rγnull, NSGTM dependent on the number of Vsa mouse); Mus musculus tandem repeats: shield hypothesis. Infect. Immun. 2004;72:6846–6851. History: A NOD-scid-gamma (NSGTM) 14. Simmons WL, Dybvig K. Biofilms mouse was presented for progressive diffuse protect Mycoplasma pulmonis cells hair loss and scaling dermatitis. (NSGTM

Presentation, NSG IL-2 Rg null mouse. The mouse has complete alopecia affecting the entire hair coat, with scaling dermatitis predominantly on the interscapular dorsum (Photo courtesy of: Unit for Laboratory Animal Medicine, In Vivo Animal Core, University of Michigan, 2800 Plymouth Road, B36-G178, Ann Arbor, MI 48109 http://animalcare.umich.edu/business-services/vivo-animal-core)

mice are typically fully haired). Four months root sheath of follicular epithelium, and prior to presentation, the mouse had sebaceous gland epithelium. Multifocal undergone chemical depletion of the native lymphocytic and macrophagic dermal hematopoietic compartment and adoptive infiltration and mild dermal fibrosis are also transfer of human CD3+-depleted bone present. The overlying epidermis has marrow mononuclear cells, followed 2 moderate diffuse orthokeratotic weeks later by human CD3+ T cell transfer. hyperkeratosis and the stratum granulosum The experimental intent was to create a is prominent (hypergranulosis). humanized hematopoietic system, which could then be used to assess effects of Other organs evaluated (not shown) experimental agents on the hematopoietic included the lungs, liver, kidneys, uterus, system. spleen, mesenteric lymph nodes, pancreas, gastrointestinal tract, and bone marrow. The Gross Pathology: The mouse had complete lungs contained diffuse peribronchial and to partial alopecia affecting the entire hair peribronchiolar lymphocytic infiltration, coat, with scaling dermatitis predominantly with occasional intraepithelial infiltrates. on the interscapular dorsum The liver and kidneys showed infrequent, perivascular and peribiliary (in liver) Laboratory results: mononuclear to lymphocytic infiltration. • PCR was performed on skin using The bone marrow, spleen, and lymph nodes primers specific for Corynebacterum were highly cellular with appropriate bovis: results were negative distribution of erythroid and myeloid precursors in marrow and red pulp and with • Aerobic culture of skin grew few - Staphylococcus aureus and rare appropriate lymphoid cellularity and Enterococcus faecium organization in the spleen and lymph nodes. Contributor’s Morphologic Diagnosis:

Microscopic Description: Skin: In sections Skin: Dermatitis, interface and adnexal, of haired skin, there is multifocal vacuolar lymphohistiocytic, with basilar vacuolar change within the basilar epidermal and change, orthokeratotic hyperkeratosis, and adnexal epithelium, evidenced by shrinking dermal fibrosis, chronic, multifocal, and separation of adjacent keratinocytes moderate with occasional intracytoplasmic vacuolation and occasional dyskeratosis. Contributor’s Comment: The gross and Shrunken cells with pyknotic nuclei, histologic findings in this case were consistent with apoptotic keratinocytes, are consistent with graft-vs-host disease multifocally present in the same areas. secondary to a protocol intended to generate Additionally, there is a mild to moderate immunologically “humanized” mice. interface and adnexal dermatitis comprised NSGTM mice2,7 have three immune-related of lymphocytes and macrophages adjacent defects, consisting of: to and infiltrating the basal epithelium, outer

1) The genetic background failure to generate mature, functional “NOD/ShiLtJ”, which is a polygenic B or T lymphocytes. defect of the innate immune system 3) A targeted null mutation of the IL2 resulting in impaired phagocytosis receptor γ chain, which blocks and defective antigen presentation by receptor recognition of cytokines IL- macrophages and dendritic cells 2, IL-4, IL-7, IL-9, IL-15, and IL-21. 2) Severe combined immunodeficiency This further impacts murine (“scid”) mutation arising from loss hematopoietic development and of function of the gene Prkdc. Prkdc results in a lack of mature, functional encodes the catalytic subunit of the NK cells (development requires IL- protein complex that controls 15). ligation of V(D)J DNA fragments during T cell receptor or Because of these broad immunologic TM immunoglobulin gene recombination defects, NSG mice are often the recipient in lymphocytes. Its absence results in strain of choice for experimental engraftment, including the transfer of human

Haired skin, NSG IL-2 Rg null mouse. Three sections of skin are presented for examination. (HE, 6X)

hematopoietic cells to generate mice with a disease.3,6 Organ specificity may relate to T “humanized” immune system.2,7 These cell homing receptor expression.1 GVHD humanized mouse protocols involve can occur acutely or have a more insidious, irradiation or chemical depletion of the chronic course. Chronic GVHD may have a native murine hematopoietic compartment more complicated pathogenesis involving and transfer of either human fetal bone autoantibody production and impaired marrow, liver, and thymus (BLT) or human development of central or peripheral bone marrow or peripheral blood-derived tolerance in graft-derived T cells. In both CD34+ stem cells. Engrafted cells migrate to acute and chronic GVHD, MHC I-mediated bone marrow and differentiate to all lineages presentation of murine antigen by graft- of the mature immune system. derived cells plays a key role, as GVHD preferentially occurs in mice receiving human grafts with specific HLA class I Humanized mouse protocols carry the antigen haplotypes, particularly those potential for development of graft-vs-host associated with autoimmune disease in 5 disease (GVHD), in which human donor T humans. GVHD in this mouse clinically cells are primed against murine antigens resembled chronic GVHD as it occurred 4 presented by graft-derived human cells, months after grafting- this is much longer generating an immune response against than the typical presentation of acute GVHD 9 murine host tissues. To avoid this, grafts are in NSG mice (3-4 weeks post-engraftment). typically depleted of mature CD3+ cells to The key feature of cutaneous GVHD is allow immature T cells, which will interface dermatitis with basilar keratinocyte ostensibly regard host tissues as “self”, to apoptosis.5 This ranges from a mild interface develop from the graft. Nevertheless, dermatitis with vacuolar change to a GVHD has been reported in humanized lichenoid (band-like) infiltration. In chronic TM NSG , most commonly in mice receiving GVHD, orthokeratotic hyperkeratosis and BLT, but occasionally in mice receiving dermal fibrosis (dermal sclerosis) become + + CD34 -selected, CD3 -depleted stem cells more prominent and inflammation may be 2-5 + grafts. Engraftment of non-CD3 - more severe, although there is considerable depleted, peripheral blood mononuclear variation and features of acute and chronic cells (PBMCs) has also been used to GVHD may be present in the same purposefully induce GVHD for study of individual.5 pathogenesis or interventional strategies.1,3 The key feature of chronic pulmonary In this mouse, the most severely affected GVHD in humans is the appearance of organs were the skin and the lung. Organs bronchiolitis obliterans, believed to result TM affected by GVHD in NSG mice resemble from chronic inflammation and fibrotic those targeted in human GVHD, with skin, remodeling of small airways. In this mouse lung, liver, gingiva, and intestinal tract case, bronchiolitis obliterans was not variably affected, depending on the type of apparent and changes were confined to a graft, the individual mouse, or the stage of

Haired skin, NSG IL-2 Rg null mouse. There is mild lymphohistiocytic interface dermatitis of the epidermis, follicles, and adnexa; and intercellular edema of the the basal epithelium. (HE 400X) ((Photo courtesy of: Unit for Laboratory Animal Medicine, In Vivo Animal Core, University of Michigan, 2800 Plymouth Road, B36-G178, Ann Arbor, MI 48109 http://animalcare.umich.edu/business-services/vivo-animal-core)

pronounced lymphohistiocytic bronchiolitis. University of Michigan It may be that fibrotic changes of the airway 2800 Plymouth Road, B36-G178 would develop with time. A recent survey of Ann Arbor, MI 48109 human patients with clinically suspected GVHD-associated bronchiolitis obliterans http://animalcare.umich.edu/business- showed that 9 of 33 biopsies showed only services/vivo-animal-core lymphocytic bronchiolitis without fibrosis – JPC Diagnosis: Haired skin: Dermatitis, this may represent an earlier stage of disease lymphohistiocytic, diffuse, mild to 5 progression. moderate, with epidermal and follicular basal cell apoptosis, intra- and extracellular Contributing Institution: edema, epidermal hyperplasia, Unit for Laboratory Animal Medicine hypergranulosis, and orthokeratotic In Vivo Animal Core hyperkeratosis.

JPC Comment: For a wide range of also shares these symptoms but appears after hematologic malignancies, a cure is only 100 days. Chronic GVHD represents 50% availble through the use of allogeneic of all cases and 25% of mortalities occurs hematologic stem cell transplants (allo- after the 100 day period. HSCT).4,7,8 Over a million of these procedures have been completed within the In humans, the rash seen in aGVHD are last decade4, in which a combination of usually centered on hair follicles, a useful 4,7 radation and chemotherapy is used to early sign for diagnosis. Histologically, eliminate neoplastic or genetically abnormal the lesion in humans is very similar to that cells of the hematopoetic compartment seen in the mouse model (and in this case), which are then replaced with hematopoetic with vacuolar degeneration of the basal stem cells from an allogeneic donor layer, dyskeratotic keratinocytes, and a mild possessing differences in human leucocyte lymphocytic infitrate at the dermo-epidermal antigens (HLA) or major (MHA) and minor junction that may infiltrate the epidermis histocompatibility antigens (miH).4 In the beginning at the rete ridges and hair 7 most simple terms, the syndrome of graft- follicles. The population of lymphocytes is + +, versus host disease results from when cells predominantly CD4 and CD8 and their from an immunocompetent donor recognize presence in the epidermis suggests that they and attack the tissues of the are donor lymphocytes attacking immunocompromised recipient (who is keratinocytes expressing different MHA 8 incapable of mounting a response against the antigens. donor’s cells). GHVD is a major cause of Chronic forms of cutaneous GVHD morbidity in allo-HSCT recipients, affecting (cGVDH) appear to be more common up to 40-60%, and also accounting for 15% overall, and are reported in between 60 and 7 of mortality. 80% of patients. The most commonly In humans, cutaneous GVHD is considered recognized forms include lichenoid GVHD the earliest and most common manifestation and sclerodermatous cGVHD. Lichenoid and may wraant a poor progosis.4,7 A cGVHD exhibits most of the histologic signs macular rash affecting a number of body descrbed about, with a macular parts, including the palms and soles, is its hyperkeratotic rash. Sclerodermatous hallmark. This condition, in addition to cGVHD may result in fibrosis at any level of 7 vomiting and jaundice resulting from small the skin and ultimately may be disfiguring. bile duct damage and cholestasis, forms the One positive aspect of GVHD is associated 8 basis for early diagnosis of acute GVHD. with an effect known as graft-versus- GVHD is divided into classic acute GVHD leukemia (GVL). It well-documented that in which symptoms appear within 100 days these patients have lower relapse rates of (often 2-3 weeks following engraftment) and hematologic malignancies, and is a useful are consistent with the triad described benefit in paitents with reduced intesity above, and late onset acute GVHD which regimens to clear the body of neoplastic hematologic cells.4

chronic graft-vs-host disease. II. The 2014 Pathology Working group Many participants had Corynebacterium report. Biol Blood Marrow bovis on ther list of differentials, but the Transplant. 2014; 21(4):589-603. moderator pointed out the lack of follicular 6. Sonntag K, Eckert F, Welker C, et hyperkeratosis, the mininal inflammation, al. Chronic graft-vs-host-disease in and the proliferation of the stratum CD34+ humanized NSGTM mice is spongiosum, rather than the stratum associated with human susceptibility granulosum, as in this case. HLA haplotypes for autoimmune References: disease. J Autoimmun. 2015; 62:55- 66. 1. Ali N, Flutter B, Sanchez Rodriguez 7. Villareal CDV, Perez JCJ, Alanis R, et al. Xenogeneic graft-vs-host JCS, Candiani JC. Cutaneous graft- disease in NOD-scid IL-2Rγnull mice versus-host disease after display a T-effector memory hematopoietic stem cell transplant – phenotype. PLoS One. 2012; a review. An Bras Dermatol 2016; 7(8):e44219. 91(3):336-343. 2. Ishikawa F, Yasukawa M, Lyons B, 8. Zeiser R, Blazar BR. Acite graft- et al. Development of functional versus-Host diseas biology, human blood and immune systems in prevention, and therapy. NOD/SCID/IL2 receptor (gamma) 9. https://www.jax.org/strain/005557 chain (null) mice. Blood. 2005; 106(5):1565-73.PMC1895228. 3. King MA, Covassin L, Brehm MA, et al. Human peripheral blood leukocyte non-obese diabetic-severe combined immunodeficiency- interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-vs-host-like disease and the role of host major histocompatibility complex. Clin Exp Immunol. 2009;157(1):104-18. 4. Rodriguez KS, Oliverira-Ribeiro C, de Abreu Fiuza Gomes S, Knobler R. Am J Clin Dermatol 2018; 19:33-50. 5. Shulman HM, Cardona DM, Greenson JK, et al. Histopathologic diagnosis of chronic graft-vs-host disease. NIH consensus development project on criteria for clinical trials in