R. S. Jayshree

Regulatory T cells in a spectrum of HPV-induced cervical lesions: cervicitis, cervical intraepithelial neoplasia and squamous cell carcinoma.

Problem  Thriving of tumors amidst rich immune infiltrates is an unexplained paradox.

ORIGINAL ARTICLE: Regulatory T Cells in a Spectrum of HPV-Induced Cervical Lesions: Cervicitis, Cervical Intraepithelial Neoplasia and Squamous Cell Carcinoma

Problem  Thriving of tumors amidst rich immune infiltrates is an unexplained paradox.

Functional tumor infiltrating TH1 and TH2 effectors in large early-stage cervical cancer are suppressed by regulatory T cells.

ObjectiveAnalysis of tumor-infiltrating lymphocytes (TILs) is one of the cornerstones for the understanding of immune responses prevailing in the tumor microenvironment. We studied TILs from squamous cell carcinoma of the cervix ex vivo without proliferating them in vitro before analysis. MethodsWhereas TILs were magnetic activated cell separation enriched and flow sorted into CD4+ CD25hi (regulatory T cells [Tregs]), CD4+ CD25int (effector T cells [Teffs]) were directly purified by flow cytometry, and both these subsets were characterized phenotypically and functionally. Tissue sections were probed for interleukin 4 (IL-4) and interferon &ggr;. ResultsEffector T cells constitutively expressed both interferon &ggr; and IL-4 prototypical cytokines of TH1 and TH2, respectively, and were able to proliferate and secrete higher quantities of both cytokines in response to anti-CD3/anti-CD28 and autologous tumor lysates. Only 53% of cervical cancer Tregs were FOXP3+, elaborated transforming growth factor &bgr;1, and IL-10 and were able to inhibit both T helper subsets. ConclusionsIntratumoral Teffs represented functionally active subsets of both TH1 and TH2 that were not anergic but were suppressed by multiple Treg subsets, which comprised FOXP3 + Tregs and Tregs secreting transforming growth factor &bgr;1 and IL-10. These results imply that the microenvironment of cervical carcinomas harbored both TH1 and TH2 subsets of CD4+ Teffs that were functionally active but were perhaps unable to perform because of the overpowering effect of Tregs.Objective Analysis of tumor-infiltrating lymphocytes (TILs) is one of the cornerstones for the understanding of immune responses prevailing in the tumor microenvironment. We studied TILs from squamous cell carcinoma of the cervix ex vivo without proliferating them in vitro before analysis. Methods Whereas TILs were magnetic activated cell separation enriched and flow sorted into CD4+ CD25hi (regulatory T cells [Tregs]), CD4+ CD25int (effector T cells [Teffs]) were directly purified by flow cytometry, and both these subsets were characterized phenotypically and functionally. Tissue sections were probed for interleukin 4 (IL-4) and interferon γ. Results Effector T cells constitutively expressed both interferon γ and IL-4 prototypical cytokines of TH1 and TH2, respectively, and were able to proliferate and secrete higher quantities of both cytokines in response to anti-CD3/anti-CD28 and autologous tumor lysates. Only 53% of cervical cancer Tregs were FOXP3+, elaborated transforming growth factor β1, and IL-10 and were able to inhibit both T helper subsets. Conclusions Intratumoral Teffs represented functionally active subsets of both TH1 and TH2 that were not anergic but were suppressed by multiple Treg subsets, which comprised FOXP3 + Tregs and Tregs secreting transforming growth factor β1 and IL-10. These results imply that the microenvironment of cervical carcinomas harbored both TH1 and TH2 subsets of CD4+ Teffs that were functionally active but were perhaps unable to perform because of the overpowering effect of Tregs.

Toll-like receptors 7, 8, and 9 expression and function in primary human cervical cancer Langerhans cells: evidence of anergy.

Objective Human papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors. Methodology Single-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a+CD207+ cells. Acute monocytic leukemia cell line THP-1–derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1β, IL-6, IL-10, IL-12p70, interferon (IFN) &agr;, interferon γ, and tumor necrosis factor (TNF) &agr; by Luminex multiplex bead array. Human papillomavirus was genotyped. Results We have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1β, and tumor necrosis factor &agr; to TLR8 ligand and interferon &agr; in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases. Conclusions Cervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

Sino-oral zygomycosis due to Absidia corymbifera in a patient with acute leukemia.

Fungi belonging to class Zygomycetes become pathogenic in certain predisposing conditions; principally diabetes mellitus, immunosuppression, trauma or burns. We report a case of a 31-year-old man with acute promyelocytic leukemia who developed infection of the sino-oral cavity, due to Absidia corymbifera during a neutropenic phase following induction chemotherapy. A provisional diagnosis of zygomycosis was made by demonstration of broad aseptate branching filamentous hyphae in the scrapings of the palate, which was subsequently confirmed as A. corymbifera by culture. Surgical debridement could not be done due to the thrombocytopenic status of the patient; instead antifungal therapy with amphotericin B was instituted. However, the patient succumbed to the infection after 15 days of its diagnosis. Although infections with Absidia are infrequent, this case highlights the need for its awareness as a potentially lethal opportunistic fungal infection that can present even with short duration of exposure to the usual risk factors.

Utility of molecular and serodiagnostic tools in cerebral toxoplasmosis with and without tuberculous meningitis in AIDS patients: A study from South India

Background: Antemortem diagnosis of cerebral toxoplasmosis, the second most common opportunistic infection (OI) in HIV-infected individuals in developing countries is a challenge. Materials and Methods: Toxoplasma gondii (T.gondii) -specific serology and nested polymerase chain reaction (nPCR) were evaluated in sera and ventricular/lumbar cerebrospinal fluid (CSF) of 22 autopsy confirmed cases of cerebral toxoplasmosis with HIV and 17 controls. Frequency of concomitant T.gondii infection was investigated in 17 cases of HIV-associated tuberculous meningitis (TBM). Results: The sensitivity, specificity, and positive and negative predictive values of T. gondii IgG on CSF (ventricular and lumbar) and sera was 100% in histology proven cerebral toxoplasmosis (concentrations: 258 ± 50, 231 ± 36, and 646 ± 243 IU/mL, respectively); majority (94%) being high avidity type, suggesting reactivation/reinfection. The sensitivity of B1 nPCR was 100% on ventricular CSF, whereas it was only 77% on lumbar CSF. Based on histology, nPCR, and IgG serology, T. gondii co-infection with TBM was observed in 65% (11/17) of cases. Discussion and Conclusion: CSF IgG serology and nPCR are tests with high sensitivity and specificity for the diagnosis of cerebral toxoplasmosis. TBM and cerebral toxoplasmosis can coexist and should be considered in the background of HIV infection in developing countries.

Cell-Free Epstein–Barr Viral Loads in Childhood Hodgkin Lymphoma: A Study from South India

Cell-free Epstein–Barr viral (EBV) DNA is detectable in plasma of patients with EBV-related lymphomas. The aim of this study was to evaluate the utility of plasma EBV DNA as a biomarker of EBV association in childhood Hodgkin lymphoma (HL). Furthermore, an attempt was made to evaluate the effectiveness of viral quantitation for assessing response to chemotherapy. Thirteen cases of childhood HL were included in this study. All 13 cases were EBV associated as reflected by expression of EBV LMP1 in the tumor specimen. Eighty-five percent had detectable EBV DNA levels; viral loads ranging from 2.9 to156.2 × 103 copies/ml (mean 29 × 103 copies/ml); while in 2 patients and 30 controls tested, viral DNA was undetectable. In four patients, follow-up samples were available after three cycles of chemotherapy; all had EBV DNAemia prior to chemotherapy but undetectable EBV DNA posttherapy. This corroborated with complete response in these four patients. Plasma EBV viral load quantification maybe a useful tool for detecting EBV association with lymphomas and in monitoring response to treatment in childhood HL in centers with limited resources, more so in India where majority of childhood HL is likely to be EBV associated. This is the first Indian study estimating plasma EBV viral loads in HL.

Oestrogen Receptor-α binds the FOXP3 promoter and modulates regulatory T-cell function in human cervical cancer

Oestrogen controls Foxp3 expression in regulatory T cells (Treg cells) via a mechanism thought to involve oestrogen receptor alpha (ERα), but the molecular basis and functional impact of ERα signalling in Treg cells remain unclear. We report that ERα ligand oestradiol (E2) is significantly increased in human cervical cancer (CxCa) tissues and tumour-infiltrating Treg cells (CD4+CD25hiCD127low), whereas blocking ERα with the antagonist ICI 182,780 abolishes FOXP3 expression and impairs the function of CxCa infiltrating Treg cells. Using a novel approach of co-immunoprecipitation with antibodies to E2 for capture, we identified binding of E2:ERα complexes to FOXP3 protein in CxCa-derived Treg cells. Chromatin immunoprecipitation analyses of male blood Treg cells revealed ERα occupancy at the FOXP3 promoter and conserved non-coding DNA elements 2 and 3. Accordingly, computational analyses of the enriched regions uncovered eight putative oestrogen response elements predicted to form a loop that can activate the FOXP3 promoter. Together, these data suggest that E2-mediated ERα signalling is critical for the sustenance of FOXP3 expression and Treg cell function in human CxCa via direct interaction of ERα with FOXP3 promoter. Overall, our work gives a molecular insight into ERα signalling and highlights a fundamental role of E2 in controlling human Treg cell physiology.

Bloodstream infections in febrile neutropenic patients at a tertiary cancer institute in South India: A timeline of clinical and microbial trends through the years.

Introduction: Febrile neutropenia (FN) is an oncological emergency. The choice of empiric therapy depends on the locally prevalent pathogens and their sensitivities, the sites of infection, and cost. The Infectious Diseases Society of America guidelines are being followed for the management of FN in India. Methods: This is a prospective observational study conducted at a tertiary care cancer centre from September 2012 to September 2014. Objectives: The objectives of this study were as follows: (1) To review the pattern of microbial flora, susceptibility pattern, and important clinical variables among bloodstream infections in febrile neutropenic patients with solid tumors and hematological malignancies. (2) As per the institutional protocol to periodically review the antibiotic policy and susceptibility pattern, and compare the findings with an earlier study done in our institute in 2010. This was a prospective study conducted from September 2012 to September 2014. Results: About 379 episodes of FN were documented among 300 patients. About 887 blood cultures were drawn. Of these, 137 (15%) isolates were cultured. Isolates having identical antibiograms obtained from a single patient during the same hospitalization were considered as one. Hence, 128 isolates were analyzed. About 74 (58%) cultures yielded Gram-negative bacilli, 51 (40%) were positive for Gram-positive cocci, and 3 (2%) grew fungi. Among Gram-negative organisms, Escherichia coli followed by Acinetobacter baumannii and Klebsiella pneumoniae accounted for 78% of the isolates. Among Gram-positive cocci, Staphylococcus species accounted for 84% of the isolates. We have noted a changing trend in the antibiotic sensitivity pattern over the years. Following the switch in empirical antibiotics, based on the results of the study done in 2010 (when the empirical antibiotics were ceftazidime + amikacin), the sensitivity to cefoperazone-sulbactam has plunged from about 80% to 60%%. Similar reduction in susceptibility was noted for piperacillin-tazobactam, imipenem, and meropenem. On the contrary, there was a marked increase in sensitivity to ceftazidime (50–76%). Based on these results, we have reverted to ceftazidime + amikacin as the empirical antibiotics. Conclusion: Every institute must have a regular revision of antibiotic policy based on periodic assessment of the clinical and microbiological profile in FN. This will combat antibiotic resistance.

Bacteraemia caused by Sciscionella marina in a lymphoma patient: phenotypically mimicking Nocardia.

A 55-year-old female patient with malignant lymphoma after induction chemotherapy developed fever. Blood culture yielded an organism biochemically identified as representing Nocardia spp., but molecular identification (16S rRNA gene sequencing) later identified it as representing Sciscionella marina. This is the first report, to the best of our knowledge, of Sciscionella being isolated from a human sample.