Lung cancer is considered one of the deadliest cancers worldwide, with a five-year survival rate of under 20% [8]. There are three different categories of NSCLC tumors: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. The most common of the tumor types is adenocarcinoma [13]. Different cell types that make up the tumor microenvironment contribute to the treatment response and prognosis of the disease. Myeloid-derived suppressor cells (MDSCs) have been shown to be increased in cancer patients compared to healthy individuals. These cells are immunosuppressive and there is evidence that MDSCs play a role in drug resistance, tumor metastases, and suppression of immune cells in cancer [3]. Antigen presenting cells (APCs) are immune cells that present antigens to B cells, macrophages, and dendritic cells. Tumors can become resistant to immunotherapy by evading T cells through modulating antigen presentation by surface MHC (major histocompatibility complex) [7]. APCs use MHC to present antigens and interact with T cells. This interaction influences T cell activation and differentiation [4]. Characterizing differences in MDSCs and APCs between normal lung samples and NSCLC samples may be helpful in identifying mechanisms of tumor progression and treatment resistance. Multiplex IHC (immunohistochemistry) is a useful technique for the identification of MDSCs and APCs and it allows for the visualization of spatial relationships between these cell types and the tumor cells.

Burkitt Lymphoma (BL) is a B cell non-Hodgkin lymphoma that occurs as three distinct subtypes, namely: endemic, sporadic, and immunodeficiency/HIV-associated. This cancer represents a frequent cause of mortality among HIV+ people in Southern Africa which has the highest incidence of HIV/AIDS worldwide. Recent reports associate a direct oncogenic function of HIV in BL development. However, the molecular mechanisms underlying this HIV-associated malignancy are not well understood. This study explores the oncogenic potential of HIV-1 protein Tat in BL via its ability to manipulate the expression of c-MYC and activation-induced cytidine deaminase (AID), two key drivers of BL progression. Using dual-luciferase reporter assays, HIV-1 Tat was shown to enhance the activity of the cMYC promoter (-2324 bp - +537 bp), which corresponded with elevated c-MYC protein levels in BL cells (Ramos) expressing HIV-1 Tat. By generating sequential promoter deletions, the minimal promoter region mediating HIV-1 Tat induced activation was identified. Site-directed mutagenesis indicated that this response was mediated by AP-1 binding elements, and coimmunoprecipitation assays revealed that HIV-1 Tat and the AP-1 factor JunB interacted within the same complex. Chromatin immunoprecipitation assays confirmed that JunB bound the c-MYC promoter in vivo under the influence of HIV-1 Tat. The effect of HIV-1 Tat on the expression of the DNA editing enzyme AID was also investigated. Dual-luciferase assays revealed that HIV-1 Tat could enhance the activity of the three regulatory regions of the AICDA gene, namely R1, R2 and R4. This translated into elevated AID protein expression in Ramos cells expressing HIV-1 Tat, which was also reflected in an increase in genomic instability as shown by enhanced phosphorylated H2AX expression. Sequential promoter deletions of the R1 promoter did not lead to a loss in HIV-1 Tat-mediated activation, pointing to potential post-transcriptional regulation. Indeed, HIV-1 Tat was found to downregulate the expression of hsa-miRNA-181b-5p, a known repressor of the murine Aicda gene. Furthermore, using reporter assays, we show that an hsa-miRNA-181b5p mimic could repress AICDA via the full-length 3'UTR which contains three putative binding elements for the miRNA. To date, this study reveals that HIV-1 Tat induced c-MYC promoter activation is mediated by two AP-1 binding sites. HIV-1 Tat was shown to couple with JunB and bind to both AP-1 sites on the c-MYC promoter inducing promoter activation. Furthermore, this study reveals a novel mechanism of AID deregulation via HIV-1 Tat-mediated miRNA perturbation. Lastly, we show that HIV-1 Tat interferes with hsa-miRNA-181b-5p expression in B cells, alleviating AICDA 3'UTR repression.

Background and aims Diagnosing lymphoma can be challenging: even in the best-resourced settings, lymphoma diagnosis may be delayed owing to the insidious onset of symptoms and/or difficulties in obtaining lymph node biopsy for diagnosis. In TB-endemic countries the diagnostic challenges in lymphoma may be further compounded by symptoms overlapping with those of TB and by resource limitations that may impede obtaining a diagnostic biopsy. New lymphoma diagnostic modalities, including the use of nextgeneration sequencing, are evolving rapidly and informing both prognosis and therapy in the field of lymphoma. These modalities of testing are likely to mean that less tissue is needed for assessment, or even that diagnosis can be made from peripheral blood. In this thesis we identify and describe local barriers in the diagnosis of lymphoma; and describe methods used to decrease the time-to-diagnosis of lymphoma and to subtype the most common lymphoma, diffuse large B-cell lymphoma (DLBCL). Our four main aims have been to: (i) Describe the pathway to a diagnosis of lymphoma in South Africa, with emphasis on the examination of local barriers to diagnosis (including overlapping symptomatology with TB) by retrospectively studying a cohort of patients with lymphoma. (ii) Investigate the diagnostic utility of the newest TB diagnostic test (the Xpert MTB/RIF Ultra); and apply this test in a diagnostic algorithm for lymphadenopathy of unknown aetiology, both on a fine-needle aspirate (FNA) from a lymph node and on tissue obtained by corebiopsy. (iii) Investigate whether a rapid-access lymph node biopsy clinic that used a core-biopsy method for lymph node biopsy was able to reduce the time-to-diagnosis of lymphoma, and to subtype accurately lymphomas for which subtyping is clinically relevant. (iv) Describe the subtype of DLBCL by HIV status using an immunohistochemical algorithm, in order both to describe the pattern of DLBCL by the most current diagnostic classification and to lay the foundations for further genetic work potentially capable of identifying mutations that could be used for genetic testing in DLBCL locally. Methods Four cohorts were analysed: (i) In the first, a group of 163 patients with lymphoma, the time-to-diagnosis and factors causing a delay in diagnosis were analysed. (ii) In the second, 99 patients with lymphadenopathy of unknown cause were recruited and the sensitivity and specificity of the Ultra were analysed using both fine-needle aspirate (FNA) and a core-biopsy technique. (iii) In the third cohort (n = 130), which included the second cohort of n = 99), outcomes from a rapid-access lymph node biopsy clinic – which used the core-biopsy method for the diagnosis of lymphoma – were analysed. (iv) In the fourth cohort (n = 182), the tissue of patients with DLBCL was analysed based on further immunohistochemical stains using the Hans algorithm in order to determine the molecular subtypes of DLBCL and determine their effect on prognosis. Results (i) In the first cohort ((n = 163), 29% HIV-infected), which was studied retrospectively, it took a median of 7 weeks for the diagnosis of lymphoma to be made from the time the patient sought medical attention. The longest time delay was in the healthcare practitioner interval (time from first healthcare visit to diagnostic biopsy). On multivariable logistic regression analysis, diagnostic delay > 6 weeks was associated with late-stage disease at presentation (odds ratio (OR) 2.3, 95% confidence interval (CI) 1.1–5.2) and a diagnosis of Hodgkin lymphoma (OR 3.0, 95% CI 1.1–8.0). HIV status was not associated with diagnostic delay (OR 0.9, 95% CI 0.3–2.2). The median time to diagnosis for patients on TB treatment was a month longer than for patients not on TB treatment. However, this was not statistically significant owing likely to a small sample size (n = 16, p = 0.28). (ii) In the second cohort, the diagnostic utility of the Ultra on lymph node aspirate and tissue were prospectively evaluated. The Ultra was found to be a sensitive and specific test for diagnosing TB of the lymph node. When compared with culture, the Ultra on aspirate had a sensitivity of 78% (40–97; 7/9) and on tissue 90% (55–100; 9/10). When using a composite reference score that combined both ‘definite TB' (culture positive) and ‘probable TB' (histological and clinical criteria), the Ultra was superior to currently used methods of diagnosing TB of the lymph node, including the detection of AFBs from an aspirate or on tissue biopsy or tissue culture (which had sensitivities of 26%, 33% and 39% respectively). The detection of granulomas on histology had high sensitivity (83%) but the lowest specificity. (iii) In the third cohort, which was studied prospectively, we obtained two important outcomes. Firstly, we showed that a rapid-access lymph node biopsy clinic was able to decrease the time-to-diagnosis of lymphoma when compared with that of cohort one (the historical cohort) to a diagnostic interval of 13.5 days compared with 48 days (p = 0.002). Secondly, the core-biopsy was able to detect lymphoma with a high rate of accuracy. The first-attempt core-biopsy was able to diagnose lymphoma in 84% of cases, and provided sufficient tissue to subtype lymphoma in a high proportion of the lymphoma cases (27/30, 90%). (iv) In the fourth, retrospectively evaluated cohort of patients with DLBCL (n = 181, 51 HIVpositive), there was a similar distribution of germinal centre (GC) and activated B cell (ABC) subtypes in the HIV-infected and HIV-uninfected groups. In contrast to what is reported in HIV-negative DLBCL literature, there were no statistically significant differences in overall survival by DLBCL subtype. Moreover, no significant difference in 5-year overall survival was demonstrated between the GCB and ABC subtypes (HR 1.2, 95% CI 0.8–1.9). Patients with HIV infection with a CD4 count of < 150 CD4 cells/mm3 had significantly poorer survival than those with no HIV infection (HR 2.4, 95% CI 1.3–4.1). Conclusions Making a diagnosis of lymphoma in South Africa is challenging. Two of the greatest challenges are overlapping symptomatology with TB and obtaining an adequate lymph node sample. The Ultra test on lymph node tissue is rapid, sensitive and specific; and can be performed on both FNA and tissue obtained through core-biopsy. Making an accurate diagnosis of TB is of critical importance, as TB is the most common cause for enlarged lymph nodes in TB-endemic areas. A reliable TB test on lymph node tissue will enable both an accurate TB diagnosis and the identification of patients who are negative for TB. The latter will require further investigation with a lymph node biopsy (via core needle or excision). As diagnostic testing and methods evolve in the direction of next-generation platforms, it is important to understand the tumour biology in our local setting, and in HIV-lymphoma, in order to be able to apply these new methods. This thesis describes the subtypes and outcomes seen in HIV DLBCL – knowledge that will be important in developing genetic next-generation sequencing methods that are specific to the type of lymphoma to be found in our context (and which may include genetic mutations induced by the Epstein–Barr virus). A clear diagnostic algorithm for the investigation of lymphadenopathy is presented, pulling together the four aims presented at the start of this section. Considering the prevalence of TB in South Africa, the goal is, in the first instance, to diagnose or exclude TB adenitis. In patients who test negative for TB, the goal then becomes to proceed with a diagnostic biopsy and, from our findings, to support the use of core-biopsy. This method, we have demonstrated, is able to provide sufficient tissue for the diagnosis and subtyping of lymphoma.

Recent acquisition of Mycobacterium tuberculosis (M.tb) infection is associated with a higher risk of tuberculosis disease, compared with remote, asymptomatic infection. M.tb infection, defined by a positive tuberculin skin test (TST) and/or IFN--release assay [IGRA e.g. QuantiFERON TB GOLD (QFT)], is commonly thought to be a chronic state. However, longitudinal studies have demonstrated the dynamic nature of M.tb infection, whereby TSTs and IGRAs revert from a positive to a negative test in some individuals, possibly an indication of bacterial clearance. Despite the first observation of discordant serial TST results over 80 years ago and the wide use of TSTs/IGRAs, there is still a limited understanding of immunological features associated with different stages of M.tb infection and discordant serial TST/IGRA results. Most studies of M.tb-specific immune responses in humans are based on cross-sectional comparisons between M.tb infection and active disease, with very few large cohort studies enabling a longitudinal assessment of different phases of infection. Thus, the main objective of this thesis was to gain a better understanding of changes in M.tbspecific CD4 T cell functional, memory and activation profiles associated with QFT conversion (acquisition of M.tb infection) and reversion (potential M.tb clearance). Our first aim was to characterise the homing, cytotoxic and functional capacity of M.tbspecific memory CD4+ T cells during recent and remote M.tb infection, with a special focus on stem cell memory T (TSCM) cells. TSCM cells play a critical role in maintaining long-lasting immunity, demonstrated by their superior longevity, proliferation and differentiation capacity compared to central memory (TCM) and effector (TE) cells. Before this study, our knowledge of TSCM cells was primarily based on virus-specific CD8+ TSCM cells. We demonstrate that M.tb-specific CD4+ TSCM cells are induced upon recent M.tb infection and maintained at steady-state during established infection. Despite being the least differentiated M.tb-specific memory subset and representing 2 years) M.tb infection, we also aimed to define an M.tb- specific T cell biomarker that can distinguish between the two infection states as current diagnostics fail to do so. Our second major finding demonstrated that recently infected individuals have lower proportions of highly differentiated IFN-+TNF+KLRG-1+ CD4+ TE cells and higher proportions of early differentiated IFN--TNF+IL2+KLRG-1- CD4+ T cells than remotely infected individuals in response to M.tb lysate but not CFP-10/ESAT-6 stimulation. Akin to their recent M.tb exposure, recently infected individuals had higher levels of T cell activation, regardless of M.tb antigen specificity, than remotely infected individuals. The degree of M.tb-specific CD4 T cell activation was identified as the best candidate biomarker for recent infection. The very same biomarker could also distinguish between progressors and non-progressors and identify individuals with active tuberculosis disease among healthy individuals with remote M.tb infection. We propose that, upon large-scale clinical validation, the T cell activation biomarker could be used as a screening test in conjunction with current tuberculosis diagnostics to guide the provision of either preventive or full tuberculosis therapy. These results have very important implications for targeting provision of preventive treatment to M.tb infected individuals at high-risk of tuberculosis, which is one of the top 10 strategies required to achieve tuberculosis elimination targets. Based on data from observational studies conducted during the pre-antibiotic era and guinea pig tuberculosis models, TST/IGRA reversion in humans is hypothesised to be associated with spontaneous (natural) clearance of infection. Similarly, individuals recently exposed to patients with tuberculosis who did not convert TST/IGRA (termed resistors) nor develop disease had M.tb-specific T cell responses that did not include IFN- production. However, clearance of M.tb infection is virtually impossible to demonstrate in healthy individuals. We clearly illustrated that acquisition infection is associated with induction of CD4+ Th1 functional and memory T cell subsets associated with increased antigen burden. We thus hypothesised that if reversion represents natural M.tb infection clearance then immune responses post-QFT reversion, if detectable, would be predominantly TSCM/TCM cells that have an IFN- independent cytokine expression profile and low T cell activation levels. Interestingly, QFT reversion was not associated with a decrease in CFP-10/ESAT-6-specific IFN-+ CD4 T cell responses detected by flow cytometry. Overall, CD4 T cell responses to CFP-10/ESAT-6 in reverters were of intermediate magnitude between non-converters and remotely infected individuals. These responses were very low in most reverters (regardless of QFT status), which may explain fluctuations around the QFT assay cut-off resulting in reversion of the test. In the reverters who had low but robustly detectable responses, CFP-10/ESAT-6-specific CD4 T cells showed low levels of M.tb-specific T cell activation, maintenance of both IFN- dependent and independent Th1 cytokine co-expressing profiles and a predominantly TTM/TE phenotype. Memory and functional profiles detected in reverters in response to M.tb lysate shared more characteristics with non-converters than persistently infected (QFT+) individuals. Based on these results, we conclude that QFT reverters represent a heterogenous population in the tuberculosis spectrum who may experience very low or no in vivo antigen exposure. Altogether, these results indicate that not everyone with a QFT+ test likely experiences ongoing in vivo M.tb exposure, as suggested by much lower T cell activation observed during remote M.tb infection and QFT reversion compared to recent M.tb infection. Whether ongoing in vivo antigen exposure is required to maintain memory responses against M.tb remains to be determined. It is possible that key features of T cell responses against M.tb, including magnitude and differentiation, are shaped by the antigen load experienced during primary infection, regardless of whether infection is subsequently cleared. Answering these questions is critical to inform the interpretation of the current immunodiagnostic assays and to determine who could be spared from preventive tuberculosis therapy. On the other hand, here we defined a biomarker of recent infection and tuberculosis disease, which could enable the provision of targeted treatment to those who would benefit the most.

We present a case of a 73-year-old male who initially presented with night sweats, intermittent fever, worsening dry cough and shortness of breath. CT scans revealed atelectasis and calcified mediastinal lymphadenopathy, raising a suspicion for sarcoidosis. Multiple lung biopsies were performed. Microscopically, atypical lymphocytes were identified within capillaries, small arteries and veins. These lymphocytes were large with prominent nucleoli. Immunohistochemical staining demonstrated tumor cells positive for CD20, CD79a, Pax-5, CD10 and Mum-1, while negative for CD3, cytokeratin, S100, and CD34. LDH serum level was increased (480 IU/L). Extra pulmonary lymphoma was not detected elsewhere in the patient. These findings support the diagnosis of primary lung intravascular large B cell lymphoma (IVLBCL). Literature review of 52 cases demonstrated occurrence of primary lung IVBCL in patients between the ages (35–85) with a slight male predominance (1.167:1). The most common clinical presentation was fever associated with dyspnea.

BACKGROUND A condyloma acuminatum is a sexually transmitted, human papillomavirus (HPV) associated, neoplasm. In men, it is predominantly found on external genitalia and rarely progresses more proximally than the distal penile urethra. Condyloma acuminata of the prostatic urethra are rare and are usually seen as an extension of, or in association with external lesions. Therefore, it is not typically considered in the differential diagnosis of isolated papillary lesions limited to the prostatic urethra. CASE REPORT A 62-year-old male with rheumatoid arthritis treated with abatacept presented to urology due to a history of intermittent bladder self-catheterization for urinary obstruction. He underwent a transurethral resection of the prostate and had incidental findings of papillary lesions restricted to the prostatic urethra that were presumed to be urothelial carcinoma. Microscopic examination established the diagnosis of condyloma acuminata, and low-risk HPV 6 and 11 were detected by in-situ hybridization. Subsequent cystoscopy showed marked growth and extension of condyloma acuminata to near the external meatus. After multiple treatments with intraurethral 5-fluorouracil, several small lesions remained in the bulbous urethra. With follow up for 2 years since diagnosis, the patient has not developed external condylomata. CONCLUSIONS A condyloma acuminatum might present as an isolated papillary growth in the prostatic urethra without clinical or historical evidence of a visible lesion on external genitalia. Immunosuppression and/or urethral instrumentation might be a risk factor for such a presentation. Urologists and pathologists should be aware of this rare possibility in order to avoid misdiagnosis, and ensure that the patient receives appropriate therapy.

No description available.

Introduction: We have observed that early transfusion with shed blood vs saline improved cerebral perfusion pressure (CPP) and cerebral venous outflow saturation (CvO2) when hypotension was superimposed on TBI. In patients, it may be difficult to transfuse within the "golden hour", which has led to searches for blood substitutes. HBOCs have shown promise in many different models, and are now in phase III clinical trials. However, the potential benefits may be outweighed by vasoconstriction due to NO scavenging. Methods: Anesthetized, ventilated swine (37±1kg) received a fluid-percussion injury and 30% hemorrhage. After 1 hr, resuscitation consisted of 500ml of Baxter's first generation HBOC (DCLHb, n=6) or saline (SAL, n=5), followed by supplemental fluid. To test cerebral vasoreactivity, FiCO 2 = 0.05 and hyperventilation were administered for 10 minutes pre- and post-resuscitation. At 72 hrs, the brains were removed for histopathology. Results: CPP (table) and arterial lactate were significantly higher following DCLHb vs. SAL resuscitation (lactate: 3.3±0.5 vs 1.2±0.2, p<0.05). CvO2changes to the CO2 challenge were similar (table). Histologic findings included subarachnoid hemorrhage, diffuse axonal injury, and infarcts. DCLHb did not aggravate these changes. Conclusions: 1) Initial resuscitation with DCLHb improved CPP but increased systemic lactate, both manifestations of systemic vasoconstriction; 2) Since there was no harmful physiologic or histologic cerebral sequela related to vasoconstriction with this first generation HBOC, second generation compounds with less NO scavenging properties may have therapeutic potential for resuscitation of TBI. CPP @ 30 min Hi CO2 - Δ CvO2 sat Lo CO2 - Δ CvO2 pre TBI post TBI pre TBI DCLHb 78±5 -4±4 -11±4 21±3 Saline 56±3 -4±2 -10±3 22±2.

Abstract A 63‐year‐old cachectic white male developed subcutaneous scalp nodules. Clinically these were felt most likely to represent metastatic tumor, although the patient was known to have rheumatoid arthritis. Aspiration cytology was interpreted as malignant due to the presence of atypical cells, but tissue biopsies demonstrated necrotizing palisading granulomas. The diagnostic hazard of cellular atypia in rheumatoid nodules is discussed.

Abstract. A case of verrucous carcinoma with a focus of cytologically malignant squamous‐cell carcinoma is presented. This usually occurs following radiation therapy of the verrucous carcinoma, but may rarely occur de novo, as in this case. The potential usefulness of fine‐needle aspiration in detecting focal anaplasia in verrucous carcinoma is discussed. This technique may be especially useful if the lesion is to be destroyed cryosurgically.