Feline immunodeficiency virus (FIV) is a lentivirus in the family Retroviridae that infects domestic cats resulting in an immunodeficiency disease featuring a progressive and profound decline in multiple sets of peripheral lymphocytes. Despite compelling evidence of FIV-associated immunopathology, there are conflicting data concerning the clinical effects of FIV infection on host morbidity and mortality. To explore FIV-associated immunopathogenesis and clinical disease, we experimentally inoculated a cohort of four specific pathogen-free kittens with a biological isolate of FIV clade C and continuously monitored these animals along with two uninfected control animals for more than thirteen years from the time of inoculation to the humane euthanasia endpoint. Here, we report the results obtained during the late asymptomatic and terminal phases of FIV infection in this group of experimentally FIV-infected cats.

Koala retrovirus is a recently endogenized retrovirus associated with the onset of neoplasia and infectious disease in koalas. There are currently twelve described KoRV subtypes (KoRV-A to I, K-M), most of which were identified through recently implemented deep sequencing methods which reveal an animals\' overall KoRV profile. This approach has primarily been carried out on wild koala populations around Australia, with few investigations into the whole-population KoRV profile of captive koala colonies to date. This study conducted deep sequencing on 64 captive koalas of known pedigree, housed in three institutions from New South Wales and South-East Queensland, to provide a detailed analysis of KoRV genetic diversity and transmission. The final dataset included 93 unique KoRV sequences and the first detection of KoRV-E within Australian koala populations. Our analysis suggests that exogenous transmission of KoRV-A, B, D, I and K primarily occurs between dam and joey. Detection of KoRV-D in a neonate sample raises the possibility of this transmission occurring in utero. Overall, the prevalence and abundance of KoRV subtypes was found to vary considerably between captive populations, likely due to their different histories of animal acquisition. Together these findings highlight the importance of KoRV profiling for captive koalas, in particular females, who play a primary role in KoRV exogenous transmission.

The role of human oncoviruses in melanoma has been poorly investigated. The aim of this study was to investigate the association between oncoviruses and melanomas searching for human papillomavirus (HPV), Epstein Barr virus (EBV), and human herpesvirus 8DNA in melanoma specimens. Formalin-fixed and paraffin-embedded tissue specimens of cutaneous, mucosal, and ocular melanomas (OM) were selected from the Pathology Departments of the Galliera Hospital (Genoa) and the University Hospitals of Turin and Cagliari. Cutaneous and mucosal nevi have been collected as controls. The oncoviruses search has been performed with different polymerase chain reaction reagent kits. Fifty-four melanomas (25 mucosal, 12 ocular, and 17 cutaneous) and 26 nevi (15 cutaneous and 11 mucosal) specimens were selected. The detection rate for one of the investigated oncoviruses was 17% in mucosal, 20% in ocular, and 0% in cutaneous melanomas (CMs). Despite the differences between groups seeming remarkable, there was no statistical significance (p > 0.5). Our data do not support a primary role of oncoviruses in melanoma carcinogenesis; however, the finding of HPV and EBV DNA in a considerable fraction of mucosal and OMs suggests that these viruses may act as cofactors in the development of extra-CMs.

Chagas cardiomyopathy (ChCM) is a severe consequence of Trypanosoma cruzi infection and has a range of electrocardiographic (ECG) and echocardiographic (ECHO) manifestations. There is a need for a standard and parsimonious research cardiac end point that does not rely on expert panel adjudication, and it is not intended to change the ChCM definition. We use data from the REDS-II cohort to propose a simplified cardiac endpoint. A total of 499 T. cruzi-seropositive blood donors were included. All participants underwent a 12-lead ECG, echocardiogram and clinical examination, and those with abnormal findings were reviewed by a panel of cardiologists who classified cases as having Chagas cardiomyopathy or not. We created an exhaustive set of ECG and ECHO finding combinations and compared these with the panel\'s classification. We selected the simplest combination that most accurately reproduced the panel\'s results. Individual ECG and ECHO variables had low sensitivity for panel-defined cardiomyopathy. The best performing combination was right bundle branch block and/or ECHO evidence of left ventricular hypocontractility. This combination had 98% specificity and 85% sensitivity for panel-defined ChCM. It was not possible to improve the overall accuracy by addition of any other ECG or ECHO variable. Substituting right bundle branch block for the more inclusive finding of QRS interval > 120 ms produced similar results. The combination of prolonged QRS interval and/or left ventricular hypocontractility closely reproduced the REDS-II expert panel classification of Chagas ChCM. In conclusion, the simple and reproducible research endpoint proposed here captures most of the spectrum of cardiac abnormalities in Chagas disease.

Follicular helper T (Tfh) cells constitute a specialized CD4+ T-cell subset that localizes in close proximity to B cells and are essential in the production of high-affinity, class-switched antibodies, and their dysregulations are also involved in autoimmune diseases. Modulating gene expression patterns in primary T cells is an important approach to understanding Tfh cell differentiation and function. In this chapter, we describe a protocol to evaluate Tfh cell differentiation with OT-II TCR transgenic T cells by retrovirally transducing gene of interest. This protocol adopts the recombinant retrovirus-based transduction of primary CD4+ T cells, and it also includes procedures for adoptive transfer, immunization, and flow cytometry analysis.

Severe congenital neutropenia (SCN) is a primary immunodeficiency disease in which a number of underlying gene defects are responsible for abnormalities in neutrophil development. The HCLS1-associated protein X1 (HAX1) mutation is associated with an autosomal-recessive form of SCN. Considering the potential of gene therapy approaches for the treatment of monogenic disorders, in this study we aimed to develop retroviral vectors expressing coding sequences (CDS) to be used for the removal of the genetic blockade in deficient hematopoietic cells. Following amplification of CDS with primers containing appropriate restriction sites, HAX1 CDS was cloned into an intermediate vector using TA-cloning. The sequence was transferred into a retroviral vector, followed by retroviral packaging in Plat-A cells. To show HAX1 protein expression, HEK293T cells were exposed to 10 multiplicity of infection (MOI) of retroviral particles and HAX1 expression was confirmed in these cells, using indirect intracellular flow cytometry. This vector was applied for in vitro transduction of hematopoietic stem cell with HAX1 mutation; after 11 days, cultured cells were analyzed for CD66acde and CD177 (neutrophil surface markers) expression. Increased neutrophil production in HAX1 viral vector-expressing hematopoietic cells was observed as compared to control vector transduced cells. Hence, according to the results, this type of therapy could be considered a potential treatment protocol for the disease.

We report two protocols to generate human pyruvate carboxylase knockdown and knockout cell lines using short hairpin RNA (shRNA) and CRISPR-Cas9 technologies. The first protocol involved cloning of a shRNA cassette targeted to human pyruvate carboxylase (PC) under the control of a U6 promoter in a retrovirus-based vector. The stable knockdown cells were achieved following infection of retroviruses expressing shRNA in target cells followed by selecting these in medium containing puromycin. The second protocol describes a CRISPR Cas9-knockout cell constructed by cloning of single guide RNA (gRNA) targeted to the human pyruvate carboxylase gene placed adjacent to Cas 9 in the pSpCas9(BB)-2A-GFP vector. The knockout cells can be selected by sorting the cells expressing GFP. We also describe protocols for detecting the level of PC mRNA and protein in the knockdown or knockout cells using qPCR and Western blot analyses, respectively. The above protocols allow investigators to create PC deficient cell lines as a tool to study role of this enzyme in cancer research.

Critical limb ischemia (CLI) is the most severe presentation of peripheral arterial disease. We developed cell-based therapy entailing intra-arterial injection of autologous venous endothelial cells (ECs) modified to express angiopoietin 1, combined with autologous venous smooth muscle cells (SMCs) modified to express vascular endothelial growth factor. This combination promoted arteriogenesis in animal models and was safe in patients with limiting claudication. In an open-label, phase Ib study, we assessed the safety and efficacy of this therapy in CLI patients who failed or were unsuitable for surgery or intravascular intervention. Of 23 patients enrolled, 18 with rest pain or non-healing ulcers (Rutherford categories 4 and 5) were treated according to protocol, and 5 with significant tissue loss (Rutherford 6) were treated under compassionate treatment. Patients were assigned randomly to receive 1 × 107 or 5 × 107 (EC-to-SMC ratio, 1:1) of the cell combination. One-year amputation-free survival rate was 72% (13/18) for Rutherford 4 and 5 patients; all 5 patients with Rutherford 6 underwent amputation. Of the 12 with unhealing ulcers at dosing, 6 had complete healing and 2 others had >66% reduction in ulcer size. Outcomes did not differ between the dose groups. No severe adverse events were observed related to the therapy.

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Helper T cell development and function must be tightly regulated to induce an appropriate immune response that eliminates specific pathogens yet prevents autoimmunity. Many approaches involving different model organisms have been utilized to understand the mechanisms controlling helper T cell development and function. However, studies using mouse models have proven to be highly informative due to the availability of genetic, cellular, and biochemical systems. One genetic approach in mice used by many labs involves retroviral transduction of primary helper T cells. This is a powerful approach due to its relative ease, making it accessible to almost any laboratory with basic skills in molecular biology and immunology. Therefore, multiple genes in wild type or mutant forms can readily be tested for function in helper T cells to understand their importance and mechanisms of action. We have optimized this approach and describe here the protocols for production of high titer retroviruses, isolation of primary murine helper T cells, and their transduction by retroviruses and differentiation toward the different helper subsets. Finally, the use of this approach is described in uncovering mechanisms utilized by microRNAs (miRNAs) to regulate pathways controlling helper T cell development and function.