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During a 3‐year time period, a 15‐year‐old male castrated Terrier mix (dog 1) and a 6‐year‐old female spayed Labrador Retriever (dog 2) presented to the North Carolina State Veterinary Hospital with similar blood work abnormalities and no significant physical examination findings. A CBC, chemistry panel, and urinalysis performed on both dogs were relatively unremarkable, other than a marked increase in serum gamma‐glutamyltransferase (GGT) activity. Through imaging, both patients were diagnosed with a renal mass, and histopathology of both masses revealed a carcinoma. Immunohistochemical staining of the renal mass in both dog 1 and dog 2 were intensely positive for GGT. Dog 1 had the affected kidney removed, which normalized the GGT value. Dog 2 was euthanized, and metastasis to the lung was noted upon postmortem examination. There have been limited case studies documenting an elevation in serum GGT in dogs diagnosed with renal carcinoma. While renal carcinoma is uncommon in dogs, it is an important differential to keep in mind when there is a marked increase in serum GGT without accompanying increases in other measured liver enzymes. In addition, serum GGT can serve as a helpful biomarker for disease resolution and recurrence, as surgical removal of the renal mass (dog 1) led to the resolution of the elevated serum GGT. To our knowledge, this is the first report demonstrating IHC staining for GGT in a canine renal carcinoma. [ABSTRACT FROM AUTHOR]

Fluid shear forces have established roles in blood vascular development and function, but whether such forces similarly influence the low-flow lymphatic system is unknown. It has been difficult to test the contribution of fluid forces in vivo because mechanical or genetic perturbations that alter flow often have direct effects on vessel growth. Here, we investigated the functional role of flow in lymphatic vessel development using mice deficient for the platelet-specific receptor C-type lectin-like receptor 2 (CLEC2) as blood backfills the lymphatic network and blocks lymph flow in these animals. CLEC2-deficient animals exhibited normal growth of the primary mesenteric lymphatic plexus but failed to form valves in these vessels or remodel them into a structured, hierarchical network. Smooth muscle cell coverage (SMC coverage) of CLEC2-deficient lymphatic vessels was both premature and excessive, a phenotype identical to that observed with loss of the lymphatic endothelial transcription factor FOXC2. In vitro evaluation of lymphatic endothelial cells (LECs) revealed that low, reversing shear stress is sufficient to induce expression of genes required for lymphatic valve development and identified GATA2 as an upstream transcriptional regulator of FOXC2 and the lymphatic valve genetic program. These studies reveal that lymph flow initiates and regulates many of the key steps in collecting lymphatic vessel maturation and development., Introduction The lymphatic vascular system regulates tissue fluid homeostasis, immune cell trafficking, and the absorption of dietary fats (1). Lymphatic capillaries lack basement membranes and maintain openings between endothelial cells [...]

Mammals transport blood through a high-pressure, closed vascular network and lymph through a low-pressure, open vascular network. These vascular networks connect at the lymphovenous (LV) junction, where lymph drains into blood and an LV valve (LVV) prevents backflow of blood into lymphatic vessels. Here we describe an essential role for platelets in preventing blood from entering the lymphatic system at the LV junction. Loss of CLEC2, a receptor that activates platelets in response to lymphatic endothelial cells, resulted in backfilling of the lymphatic network with blood from the thoracic duct (TD) in both neonatal and mature mice. Fibrin-containing platelet thrombi were observed at the LVV and in the terminal TD in wild-type mice, but not Clec2-deficient mice. Analysis of mice lacking LVVs or lymphatic valves revealed that platelet-mediated thrombus formation limits LV backflow under conditions of impaired valve function. Examination of mice lacking integrin-mediated platelet aggregation indicated that platelet aggregation stabilizes thrombi that form in the lymphatic vascular environment to prevent retrograde blood flow. Collectively, these studies unveil a newly recognized form of hemostasis that functions with the LVV to safeguard the lymphatic vascular network throughout life., Introduction The mammalian cardiovascular system is divided into distinct blood and lymphatic vascular networks: blood is transported to the tissues in a closed, high-pressure vascular system, and interstitial fluid is [...]

A transmembrane O-glycoprotein podoplanin (PDPN) expressed on fibroblastic reticular cells is the activating ligand for platelet receptor CLEC-2; this interaction leads to perivenular release of sphingosine-1-phosphate and expression of VE-cadherin on high endothelial venules, a key process for the maintenance of vascular integrity in lymph nodes. Role of podoplanin in lymph node homeostasis The lymph nodes, essential sites for immune responses, are supplied with circulating lymphocytes that enter via specialized post-capillary blood vessels known as high endothelial venules (HEVs). How the integrity of this vascular barrier is maintained in the face of the increase in lymphocyte homing during immune responses is not known. Lijun Xia and colleagues now show that the mechanism involves cross-talk between HEVs, platelets and the fibroblastic reticular cells (FRCs) that surround the HEVs. The transmembrane protein podoplanin, expressed on FRCs, interacts with the platelet activation receptor CLEC-2, prompting the release of sphingosine-1-phosphate that in turn preserves the adhesion molecule VE-cadherin on the HEVs. This finding may suggest new approaches to the regulation of lymph node homeostasis in conditions of increased HEV proliferation, such as chronic inflammation. Circulating lymphocytes continuously enter lymph nodes for immune surveillance through specialized blood vessels named high endothelial venules.sup.1,2,3,4,5, a process that increases markedly during immune responses. How high endothelial venules (HEVs) permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1[alpha]).sup.6,7,8 in maintaining HEV barrier function. Mice with postnatal deletion of Pdpn lost HEV integrity and exhibited spontaneous bleeding in mucosal lymph nodes, and bleeding in the draining peripheral lymph nodes after immunization. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells.sup.7, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2, also known as CLEC1B).sup.9,10. Mice lacking fibroblastic reticular cell PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (also known as CDH5), which is essential for overall vascular integrity.sup.11,12, on HEVs. Infusion of wild-type platelets restored HEV integrity in Clec-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate.sup.13,14 from platelets, which promoted expression of VE-cadherin on HEVs ex vivo. Furthermore, draining peripheral lymph nodes of immunized mice lacking sphingosine-1-phosphate had impaired HEV integrity similar to Pdpn- and Clec-2-deficient mice. These data demonstrate that local sphingosine-1-phosphate release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses., Author(s): Brett H. Herzog [sup.1] [sup.2] , Jianxin Fu [sup.1] [sup.3] [sup.4] , Stephen J. Wilson [sup.5] , Paul R. Hess [sup.6] , Aslihan Sen [sup.6] , J. Michael McDaniel [...]

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway., Introduction Platelets play an essential role in the prevention of excessive blood loss after injury (hemostasis), but can also form occlusive thrombi at sites of atherosclerotic plaque rupture (thrombosis) (1). [...]

Canine spontaneous cancers may offer greater fidelity than rodent models in advancing clinical immunotherapies. Boxers in particular are distinguished as study subjects by their popularity, and high incidence of human‐relevant cancers. Further, the MHC class I allele DLA‐88*034:01, with a known motif, dominates the breed, facilitating discovery of shared CTL responses against mutation‐origin neoepitopes by standard prediction methods. We experimentally confirmed the allomorph's binding motif by developing an MHC surface stabilization assay. The assay validated four DLA‐88*034:01‐presented peptides from canine distemper virus, ubiquitously administered in routine vaccines, for positive controls in future CTL studies. In turn, these viral peptides substantiated motif‐based prediction for DLA‐88*034:01. The study adds new tools for studying neoepitope‐specific CTL in Boxers to foster canine comparative oncology. [ABSTRACT FROM AUTHOR]

Cancer‐testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies, mostly solid tumours, due to epigenetic de‐repression. Normally expressed only in fetal or gametogenic tissues, CTAs are tantalizing immunotherapy targets, since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers, and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this, the ligandome binding the dog leukocyte antigen (DLA)‐88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA‐derived peptides, which could serve as CD8+ T‐cell epitopes. Twenty‐two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumour and normal tissue by quantitative and end‐point RT‐PCR. The ortholog of an established CTA, IGF2BP3, had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis‐enhanced proteins were also assessed. AKR1E2, SPECC1 and TPX2 were expressed variably in HS and T‐cell lymphoma biopsies, but also at high levels in critical tissues, including kidney, brain and marrow, diminishing their utility. A more tissue‐restricted candidate, NT5C1B, was detected in T‐cell lymphomas, but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach, proceeding from identification of MHC class I‐presented peptides to a comparative RNA expression survey of tumours and normal tissues. [ABSTRACT FROM AUTHOR]

Background: Doxorubicin (DOX) can cause cumulative cardiotoxicity in dogs, but the incidence of clinical cardiotoxicity in dogs receiving DOX has not been determined. Hypothesis/Objectives: To determine if the duration of DOX infusion influences the incidence of cardiotoxicity, to characterize the incidence of clinical cardiotoxicity in dogs during or after DOX chemotherapy, and to identify any risk factors associated with cardiotoxicity. Animals: Four‐hundred ninety‐four dogs that received at least 1 dose of DOX for the treatment of cancer. Methods: Retrospective study of dogs that received DOX from 2006 to 2015. Results: Of 494 dogs, 20 (4.0%) developed clinical cardiotoxicity. The duration of DOX infusion was not significantly associated with clinical cardiotoxicity, whereas a higher cumulative dose of DOX, higher body weight, decreases in fractional shortening after 5 doses of DOX, and development of ventricular premature contractions were significantly associated with clinical cardiotoxicity. High‐risk breeds for developing dilated cardiomyopathy had an incidence of 15.4%, whereas low‐risk breeds had an incidence of 3.0%. Conclusions and Clinical Importance: Although the duration of DOX infusion did not influence the incidence of cardiotoxicity, premature contractions and decreases in fractional shortening should raise concern for the development of clinical cardiotoxicity. Overall, the incidence of clinical DOX‐induced cardiotoxicity is low, but Boxers and other breeds at high risk for dilated cardiomyopathy may be at an increased risk. [ABSTRACT FROM AUTHOR]

Development of effective immunotherapy for chemoresistant malignancies can be advanced by studies in spontaneous cancer models, such as the dog. A crucial first step, T‐cell epitope discovery, can be assisted by determination of binding motifs of common dog leukocyte antigen (DLA) class Ia allotypes. Boxers are popular, inbred dogs with increased risks of relevant target cancers and restricted MHC diversity. We sought to identify the motif of DLA‐88*034:01, a breed‐dominant allotype, to assist peptide prediction from tumor antigens. Mass spectrometry of eluted peptides showed a preference for nonamers with conserved amino acid preferences: basic at position (P)1; hydrophobic at P2; acidic at P4; histidine at P6; and phenylalanine at P9. This data should expedite finding epitopes restricted by this DLA‐88 allotype. [ABSTRACT FROM AUTHOR]

Summary: The JAK2 V617F mutation is characteristic of most Philadelphia chromosome‐negative myeloproliferative neoplasms (MPNs) and occurs rarely in de novo acute myeloid leukaemia (AML). We sought to characterize AMLs that harbour this mutation and distinguish those that arise de novo (AML‐DN) from those that reflect transformation of an underlying MPN (AML‐MPN). Forty‐five patients with JAK2 V617F‐mutated AML were identified; 15 were AML‐DN and 30 were AML‐MPN. AML‐MPN cases were more likely to have splenomegaly (P = 0·02), MPN‐like megakaryocytes and higher mean JAK2 V617F VAF at diagnosis (P = 0·04). Mutations involving TET2 were exclusively identified in AML‐DN patients. Mutations of genes affecting DNA methylation were more common in AML‐DN (P < 0·01). A complex karyotype was more frequent in AML‐MPN cases than in AML‐DN (P < 0·01), with AML‐DN more likely to display a normal karyotype (P = 0·02). Bone marrow histology after recovery from induction chemotherapy in AML‐DN cases revealed no morphological evidence of any previously occult MPNs, while this was evident in most of the AML‐MPN specimens (P < 0·01). These findings in this largest study of JAK2 V617F‐mutated AMLs indicate that AML‐DN is distinct from AML‐MPN. [ABSTRACT FROM AUTHOR]

Antigen-specific T-cells are highly variable, spanning potent antiviral efficacy and damaging auto-reactivity. In virus infections, identifying the most efficacious responses is critical to vaccine design. However, current methods depend on indirect measures or on ex vivo expanded CTL clones. We here describe a novel application of cytotoxic saporin-conjugated tetramers to kill antigen-specific T-cells without significant off-target effects. The relative efficacy of distinct antiviral CD8+ T-cell specificity can be directly assessed via antigen-specific CD8+ T-cell depletion. The utility of these reagents is demonstrated here in identifying the CD8+ T-cell specificity most effective in preventing HIV progression in HIV-infected HLA-B*27-positive immune controllers. [ABSTRACT FROM AUTHOR]

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1+, c-Kit+ (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6-18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC. [ABSTRACT FROM AUTHOR]

Islet-infiltrating lymphocytes of individual male and female non-obese diabetic (NOD) mice were examined with the purpose of determining the differences that lead to a predominance of diabetes in female versus males NOD mice. When normalized for the amount of islet lymphocytes recovered, the infiltrating lymphocytes of female NOD mice were indistinguishable from those of male NOD mice. The only observed difference was that islet inflammation progressed at an increased rate in female compared to male NOD mice. There was no difference in the composition of islet infiltrates in male and female NOD mice. Unexpectedly, the ratio of CD4+:CD8+ T cells was tightly controlled in the islets throughout diabetogenesis. The frequency of IL-4+ CD4+ T cells started high but quickly fell to 3% of the population that was maintained with increasing inflammation. A significant portion of the CD8+ T cells were islet-specific glucose-6-phosphatase catalytic subunit-related protein specific in both male and female NOD mice and this population was antigen experienced and increased at high levels of islet inflammation. Surprisingly, a large pool of antigen inexperienced naïve T cells was detected in the islets. We conclude the underlying immunological processes in both male and female NOD mice are similar while the rates differ and the presence of naïve T cell in the islets may contribute to epitope spreading. [ABSTRACT FROM AUTHOR]

OBJECTIVE To evaluate the efficacy of doxorubicin for treatment of histiocytic sarcoma (HS) in dogs, whether administered as the sole treatment or as an adjunct to surgery or radiation therapy. ANIMALS 31 client-owned dogs with localized or disseminated HS examined between 2003 and 2017. PROCEDURES Medical records were reviewed retrospectively, and data were collected. The Kaplan-Meier method was used to estimate time-to-progression from the date of first doxorubicin administration and survival time from initial diagnosis. Factors that could be associated with poorer outcomes with doxorubicin treatment were analyzed with log-rank tests. RESULTS The objective response rate (ORR) was 26%. When stratified by disease status, dogs with localized and disseminated forms experienced 43% and 21% ORRs, respectively. Median time to progression after initiating doxorubicin treatment (n = 30 dogs) was 42 days. Median survival time from initial diagnosis to death (n = 29 dogs) was 169 days. Complete responses were obtained in only 2 dogs that had localized disease and received multimodality therapy. CLINICALRELEVANCE Benefits of doxorubicin administration in canine HS are modest, with a limited ORR and delay in tumor progression, and are comparable to effects attained with other single-agent regimens. [ABSTRACT FROM AUTHOR]

Objective--To evaluate the usefulness of a PCR-based telomeric repeat amplification protocol (TRAP) assay for detecting telomerase activity in cells from a canine transitional cell carcinoma (TCC) cell line and, ultimately, in the urine of dogs with TCC. Animals--11 dogs with histologic or cytologic evidence of TCC, 10 dogs with benign lower urinary tract disease, and 9 healthy dogs. Procedures--Telomerase activity was initially evaluated in cells from canine TCC (K9TCC) and telomerase-negative (WI-38) cell lines. Following assay optimization, telomerase stability was evaluated at various storage durations and temperatures. Urine samples were then obtained prospectively from study dogs. Results--Telomerase activity was detected in the K9TCC cell line. The TRAP assay detected telomerase activity in as few as 10 K9TCC cells alone and as low as 2% of a total cell population in K9TCC and WI-38 mixing experiments. A loss of telomerase activity was detected with increasing urine storage durations at various temperatures. Telomerase activity was clearly detected in samples collected from 10 of 11 dogs with TCC, 2 of 10 dogs with benign lower urinary tract disease, and none of the 9 healthy dogs. Conclusions and Clinical Relevance--The TRAP-based assay detected telomerase activity in the canine TCC cell line and revealed that the telomerase ribonucleoprotein complex was inherently unstable at various storage durations and conditions. Telomerase activity was also detectable in urine samples obtained from dogs with TCC, which suggested the TRAP assay may be useful in diagnosing TCC in dogs. [ABSTRACT FROM AUTHOR]

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-Db-restricted, immunodominant T-cell responses by deleting precursor CTL Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that Db-Uty+ and Db-Smcy+ T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8+ T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation. [ABSTRACT FROM AUTHOR]

Objective--To determine the frequency of clinically relevant abnormalities missed by fail-ure to perform a blood smear evaluation in a specific subset of dogs receiving chemo-therapy and to compare automated and manual neutrophil counts in the same population. Design--Retrospective case series. Animals--50 dogs receiving chemotherapy with a total nucleated cell count > 4,000 nucle-ated cells/μL. Procedures--50 blood smears were evaluated for abnormalities that have strong potential to change the medical plan for a patient: presence of blast cells, band neutrophils, nucle-ated RBCs, toxic change, hemoparasites, schistocytes, and spherocytes. Automated and manual neutrophil counts were compared. Results--Blood smears from 10 (20%) patients had > 1 abnormalities. Blast cells were identified on 4 (8%) blood smears, increased nucleated RBCs were identified on 5 (10%), and very mild toxic change was identified on 2 (4%). Correlation coefficient of the neutro-phil counts was 0.96. Analysis revealed a slight bias between the automated and manual neutrophil counts (mean ± SD difference, -0.43 X 10³/μL ± 1.10 X 10³/μL). Conclusions and Clinical Relevance--In this series of patients, neutrophil count corre-lation was very good. Clinically relevant abnormalities were found on 20% of the blood smears. An automated CBC appears to be accurate for neutrophil counts, but a microscopic examination of the corresponding blood smear is still recommended; further studies are needed to determine whether the detection or frequency of these abnormalities would dif-fer dependent on chemotherapy protocol, neoplastic disease, and decision thresholds used by the oncologist in the ordering of a CBC without a blood smear evaluation. [ABSTRACT FROM AUTHOR]

Objective--To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM)in dogs. Animals--111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which Iocoregional disease control was achieved. Procedures--58 dogs received an initial series of 4 injections of huTyr vaccine (102 pg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death. Results--Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising. Conclusions and Clinical Relevance--Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM. Impact for Human Medicine--Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease. [ABSTRACT FROM AUTHOR]

T-cell production depends on the recruitment of hematopoietic progenitors into the thymus. T cells are among the last of the hematopoietic lineages to recover after bone marrow transplantation (BMT), but the reasons for this delay are not well understood. Under normal physiologic conditions, thymic settling is selective and either CCR7 or CCR9 is required for progenitor access into the thymus. The mechanisms of early thymic reconstitution after BMT, however, are unknown. Here we report that thymic settling is briefly CCR7/CCR9-independent after BMT but continues to rely on the selectin ligand PSGL-1. The CCR7/CCR9 independence is transient, and by 3 weeks after BMT these receptors are again strictly required. Despite the normalization of thymic settling signals, the rare bone marrow progenitors that can efficiently repopulate the thymus are poorly reconstituted for at least 4 weeks after BMT. Consistent with reduced progenitor input to the thymus, intrathymic progenitor niches remain unsaturated for at least 10 weeks after BMT. Finally, we show that thymic recovery is limited by the number of progenitors entering the thymus after BMT. Hence, T-lineage reconstitution after BMT is limited by progenitor supply to the thymus. [ABSTRACT FROM AUTHOR]

The scent detection prowess of dogs has prompted interest in their ability to detect cancer. The purpose of this study was to determine whether dogs could use olfactory cues to discriminate urine samples collected from dogs that did or did not have urinary tract transitional cell carcinoma (TCC), at a rate greater than chance. Dogs with previous scent training (n=4) were initially trained to distinguish between a single control and a single TCC-positive urine sample. All dogs acquired this task (mean =15±7.9 sessions; 20 trials/session). The next training phase used four additional control urine samples (n=5) while maintaining the one original TCC-positive urine sample. All dogs quickly acquired this task (mean =5.3±1.5 sessions). The last training phase used multiple control (n=4) and TCC-positive (n=6) urine samples to pro-mote categorical training by the dogs. Only one dog was able to correctly distinguish multiple combinations of TCC-positive and control urine samples suggesting that it mastered categorical learning. The final study phase evaluated whether this dog would generalize this behavior to novel urine samples. However, during double-blind tests using two novel TCC-positive and six novel TCC-negative urine samples, this dog did not indicate canine TCC-positive cancer samples more frequently than expected by chance. Our study illustrates the need to consider canine olfactory memory and the use of double-blind methods to avoid erroneous conclusions regarding the ability of dogs to alert on specimens from canine cancer patients. Our results also suggest that sample storage, confounding odors, and other factors need to be considered in the design of future studies that evaluate the detection of canine cancers by scent detection dogs., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Mammals must inflate their lungs and breathe within minutes of birth to survive. A key regulator of neonatal lung inflation is pulmonary surfactant, a lipoprotein complex which increases lung compliance by reducing alveolar surface tension (Morgan, 1971). Whether other developmental processes also alter lung mechanics in preparation for birth is unknown. We identify prenatal lymphatic function as an unexpected requirement for neonatal lung inflation and respiration. Mice lacking lymphatic vessels, due either to loss of the lymphangiogenic factor CCBE1 or VEGFR3 function, appear cyanotic and die shortly after birth due to failure of lung inflation. Failure of lung inflation is not due to reduced surfactant levels or altered development of the lung but is associated with an elevated wet/dry ratio consistent with edema. Embryonic studies reveal active lymphatic function in the late gestation lung, and significantly reduced total lung compliance in late gestation embryos that lack lymphatics. These findings reveal that lymphatic vascular function plays a previously unrecognized mechanical role in the developing lung that prepares it for inflation at birth. They explain respiratory failure in infants with congenital pulmonary lymphangiectasia, and suggest that inadequate late gestation lymphatic function may also contribute to respiratory failure in premature infants.

Major histocompatibility complex (MHC) class I and MHC class II molecules present short peptides that are derived from endogenous and exogenous proteins, respectively, to cognate T-cell receptors (TCRs) on the surface of T cells. The exquisite specificity with which T cells recognize particular peptide-major-histocompatibility-complex (pMHC) combinations has permitted development of soluble pMHC multimers that bind exclusively to selected T-cell populations. Because the pathogenesis of type 1 diabetes mellitus (T1DM) is driven largely by islet-reactive T-cell activity that causes β-cell death, these reagents are useful tools for studying and, potentially, for treating this disease. When coupled to fluorophores or paramagnetic nanoparticles, pMHC multimers have been used to visualize the expansion and islet invasion of T-cell effectors during diabetogenesis. Administration of pMHC multimers to mice has been shown to modulate T-cell responses by signaling through the TCR or by delivering a toxic moiety that deletes the targeted T cell. In the nonobese diabetic mouse model of T1DM, a pMHC-I tetramer coupled to a potent ribosome-inactivating toxin caused long-term elimination of a specific diabetogenic cluster of differentiation 8+ T-cell population from the pancreatic islets and delayed the onset of diabetes. This review will provide an overview of the development and use of pMHC multimers, particularly in T1DM, and describe the therapeutic promise these reagents have as an antigen-specific means of ameliorating deleterious T-cell responses in this autoimmune disease., (© 2012 Diabetes Technology Society.)

The field of platelet biology has rapidly expanded beyond the classical role of platelets in preventing blood loss and orchestrating clot formation. Despite the lack of transcriptional ability of these anuclear cell fragments, platelet function is now thought to encompass such diverse contexts as tissue repair, immune activation, primary tumor formation, and metastasis. Recent studies from multiple groups have turned the spotlight on an exciting new role for platelets in the formation of lymphatic vessels during embryonic development. Genetic experiments demonstrate that podoplanin, a transmembrane protein expressed on lymphatic endothelial cells, engages the platelet C-type lectin-like receptor 2 (CLEC-2) when exposed to blood, leading to SYK-SLP-76-dependent platelet activation. When components of this pathway are disrupted, aberrant vascular connections form, resulting in blood-lymphatic mixing. Furthermore, platelet-null embryos manifest identical blood-lymphatic mixing. The identification of platelets as the critical cell type mediating blood-lymphatic vascular separation raises new questions in our understanding of lymphatic development and platelet biology.

There is compelling evidence that self-reactive CD8(+) T cells are a major factor in development and progression of type 1 diabetes in animals and humans. Hence, great effort has been expended to define the specificity of autoimmune CD8(+) T cells and to alter their responses. Much work has focused on tolerization of T cells using proteins or peptides. A weakness in this approach is that residual autoreactive T cells may be activated and exacerbate disease. In this report, we use a novel approach, toxin-coupled MHC class I tetramers. Used for some time to identify Ag-specific cells, in this study, we use that same property to delete the Ag-specific cells. We show that saporin-coupled tetramers can delete islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-reactive T cells in vitro and in vivo. Sequence analysis of TCRbeta-chains of IGRP(+) cells reveals the repertoire complexity in the islets is markedly decreased as NOD mice age and significantly altered in toxic tetramer-treated NOD mice. Further tetramer(+) T cells in the islets are almost completely deleted, and, surprisingly, loss of tetramer(+) T cells in the islets is long lasting. Finally, we show deletion at 8 wk of age of IGRP(+) CD8(+) T cells, but not dystophia myotonica kinase- or insulin B-reactive cells, significantly delays diabetes in NOD mice.

CD8+ cytotoxic T lymphocytes (CTLs) are important effector cells responsible for tissue destruction in several autoimmune and allograft-related diseases. To discover if pathogenic T cells could be selectively deleted, we investigated the ability of a toxin coupled to major histocompatibility complex (MHC) class I tetramers to kill antigen-specific CD8+ T cells. H2-D(b) tetramers were assembled using streptavidin conjugated to the ribosome-inactivating protein (RIP) saporin (SAP). These tetramers inhibited ribosome activity in vitro, retained the T-cell receptor (TCR)-binding specificity of their nontoxic counterparts, and were internalized by 100% of target cells, leading to cell death in 72 hours. Cytotoxicity was dependent on the tetramer dose and avidity for the T cell. A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact.