Cryo-EM structures of perforin-2 in isolation and assembled on a membrane suggest a mechanism for pore formation.

Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 ß-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.

Co-Occurrence of Familial Hemophagocytic Lymphohistiocytosis Type 2 and Chronic Active Epstein-Barr Virus in Adulthood.

We report, to the best of our best knowledge, the oldest individual to ever be diagnosed with Familial Hemophagocytic Lymphohistiocytosis (FHL) Type 2 from homozygous c.1349C>T (p.T450M) missense variants in the PRF1 gene. This rare case advanced in complexity with a simultaneous diagnosis of Chronic Active Epstein-Barr Virus (CAEBV) - a distinct clinical entity from acute EBV infections and a well-described trigger of Hemophagocytic Lymphohistiocytosis (HLH). This is, to the best of our knowledge, the only individual to ever be diagnosed with CAEBV in the setting of this specific variant and the oldest to be diagnosed with a coexisting perforin variant. This case provides understanding of EBV, human genetics, and lymphoproliferative disorders while adding a unique differential diagnosis to adults who present with fever of unknown origin and diffuse lymphadenopathy without evidence of malignancy. This report explores the diagnosis and treatment of both HLH and CAEBV, encouraging discussion regarding current clinical management and future research needs.

Immunohistochemical study of perforin and apoptosis stimulation fragment ligand (FasL)in active vitiligo.

Several studies demonstrated a major pathological role of melanocyte-specific cytotoxic CD8+ T cells in the pathogenesis of vitiligo. It has been suggested that apoptosis, rather than necrosis, is the mechanism of melanocyte depletion in vitiligo. The aim of this study was to evaluate the expression and distribution of perforin and apoptosis stimulation fragment ligand (FasL) in the epidermis and dermis of the perilesional and non-lesional skin of vitiligo patients in comparison to controls, to assess their possible role in mediating apoptosis in vitiligo. Twenty patients with active non-segmental vitiligo and 20 healthy controls were enrolled in the study. Skin biopsies were taken from perilesional and non-lesional skin of patients with vitiligo, as well as covered skin of controls. Immunostaining for perforin and FasL was performed and the quantitative analysis for the expression of perforin and FasL was carried out in the epidermis and dermis of biopsied specimens. Epidermal perforin, dermal perforin, epidermal FasL, dermal FasL were significantly higher in perilesional as well as non-lesional skin than controls. There was a statistically significant positive correlation between epidermal and dermal perforin in perilesional skin. There was a statistically significant positive correlation between epidermal and dermal perforin, as well as epidermal and dermal FasL in non-lesional skin. In conclusion, the significant expression of perforin and FasL in the epidermis and dermis of both perilesional and non-lesional skin of active vitiligo patients suggests the role of cytotoxic granules and apoptotic cell death pathways in the pathogenesis of active vitiligo.

Diagnostic performance of culture filtered protein 10-specific perforin in pediatric patients with active tuberculosis.

BACKGROUND: Mycobacterium tuberculosis (Mtb)-specific perforin were significantly increased in patients with tuberculosis. This study aims to evaluate the diagnosis value of Mtb-specific perforin in pediatric patients with tuberculosis. METHODS: Diagnostic performance of perforin levels induced by 6-kDa early secreted antigen target (ESAT6) or culture filtered protein 10 (CFP10) were evaluated in eighty-six samples from children participants by receiver operating characteristic curve analysis. Flow cytometry was used to detect the expression of perforin and INF-γ of CD4+ , CD8+ T cells in response to CFP10 stimulation. RESULTS: After ex vivo stimulation, levels of ESAT6/CFP10-specific perforin in LTBI patients were significantly higher than active TB (ATB) patients, non-tuberculosis infection (non-TB), and health control (HC) individuals. The diagnostic efficacy of CFP10-specific perforin for TB diagnosis was significantly higher than ESAT6-specific perforin and T-SPOT assay, and when 0.74 ng/mL was taken as the cutoff value, the sensitivity, specificity, and accuracy were 97.83%, 87.5%, and 93.02%. CFP10-specific perforin in both CD4+ and CD8+ T cells were significantly higher in ATB patients compared to HCs and further increased in LTBI patients. However, INF-γ was mainly secreted by CD4+ T cells and showed no significant difference between LTBI and ATB patients. In addition, CFP10-specific perforin can effectively distinguish between ATB and LTBI with the cutoff value of 1.80 ng/mL. Sensitivity and specificity were 88.46% and 85.62%, respectively. CONCLUSIONS: CFP10-specific perforin may be used as a novel cellular immunity-based diagnostic marker of pediatric patients with tuberculosis, and with the potential for discriminating ATB from LTBI.

Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms.

Immune therapy of cancer is among the most promising recent advances in medicine. Whether the immune system can keep cancer in check depends on, among other factors, the efficiency of immune cells to recognize and eliminate cancer cells. We describe a time-resolved single-cell assay that reports the quality, quantity, and kinetics of target cell death induced by single primary human natural killer (NK) cells. The assay reveals that single NK cells induce cancer cell death by apoptosis and necrosis but also by mixed forms. Inhibition of either one of the two major cytotoxic pathways, perforin/granzyme release or FasL/FasR interaction, unmasked the parallel activity of the other one. Ca2+ influx through Orai channels is important for tuning killer cell function. We found that the apoptosis/necrosis ratio of cancer cell death by NK cells is controlled by the magnitude of Ca2+ entry and furthermore by the relative concentrations of perforin and granzyme B. The possibility to change the apoptosis/necrosis ratio employed by NK cells offers an intriguing possibility to modulate the immunogenicity of the tumor microenvironment.

Decrease of perforin positive CD3+γδ-T cells in patients with obstructive sleep disordered breathing.

INTRODUCTION: Sleep related breathing disorders (SRBD) cause sleep fragmentation, intermittent hypoxia or a combination of both leading to homeostasis perturbations, including in the immune system. We investigated whether SRBD patients with or without intermittent hypoxia show substantial differences in perforin and granzyme-B positive peripheral blood lymphocytes. METHODS: A total of 87 subjects were included and distributed as follows: 24 controls (C), 19 patients with respiratory effort related arousals due to increased upper airway resistance (UAR) without hypoxic events, 24 obese patients with obstructive sleep apnea (OSA) (oOSA), and 20 without obesity (noOSA). After polysomnographic recording, we analyzed in fasting blood samples routine hematologic and biochemical parameters and the percentage of lymphocytes containing the proteins perforin and granzyme-B (GrB). Kruskal-Wallis tests and a posteriori multiple comparisons were applied for statistical analysis of results. RESULTS: Perforin-positive γδ-cells revealed significant differences between groups (p = 0.017), especially between the Control group and the oOSA (p-value = 0.04); the remaining SRBD groups also showed differences from the control (C vs UAR: p = 0.08; C vs noOSA = 0.09), but they did not raise to statistical significance. There were no differences among the SRBD groups. Granzyme-B cells were decreased in SRBD patients, but the differences were not statistically significant. No additional statistical significant result was found in the other investigated lymphocyte subsets. CONCLUSIONS: Obstructive sleep-disordered breathing is associated with a decrease in perforin-positive CD3+γδ-T cells. Although this finding was detected in lean patients without intermittent hypoxia, the reduction was only statistically significant in obese patients with severe OSA. Because CD3+γδ-T cells play an important role in the control of tumor cells, our findings are directly relevant for the study of the association of OSA and cancer.

Frequency and perforin expression of different lymphocyte subpopulations in patients with lower limb fracture and thoracic injury.

INTRODUCTION: Trauma with multiple injuries is associated with a high risk of complications, which may be related to excessive stimulation of inflammatory and anti-inflammatory responses. Although the effects of polytrauma on the immune response have been well established at the cellular and molecular levels, there is little information about the changes in the cytolytic potential of immunocompetent cells, including expression of cytotoxic molecules such as perforin. Therefore, the objective of the present study was to analyse and compare differences in the frequency and perforin expression of leukocyte subpopulations in the peripheral blood of patients with lower limb fracture, thoracic injury, and simultaneous lower limb fracture and thoracic injury. PATIENTS AND METHODS: Forty-five patients with trauma injury (15 patients with lower limb injury, 15 patients with thoracic injury, and 15 patients with simultaneous lower limb and thoracic injury) were included in the study. Peripheral blood of 15 sex- and age-matched healthy volunteers served as the control group. Peripheral blood samples were taken from all subjects included in the study and peripheral blood mononuclear cells were isolated by gradient centrifugation. The frequency of T lymphocytes, natural killer (NK) and NK T cells, and their subsets, as well as their perforin expression levels were simultaneously detected and analysed by flow cytometry. RESULTS: There was a statistically significant decrease in the frequency of T lymphocytes, NK and NK T cells as well as perforin expression in the patients with simultaneous lower limb and thoracic injury compared with the other two groups, with a predominantly marked decrease in NK and NK T cells. CONCLUSION: The decrease in the frequency and cytotoxic potential of peripheral blood lymphocytes is related to the severity of trauma injury, which can explain the underlying mechanism contributing to complication occurrence.

High Perforin-Positive Cardiac Cell Infiltration and Male Sex Predict Adverse Long-Term Mortality in Patients With Inflammatory Cardiomyopathy.

BACKGROUND: The authors analyzed the effects of perforin-dependent infiltration on long-term mortality in patients with inflammatory cardiomyopathy (CMi). We previously demonstrated that left ventricular function deteriorates and progresses to substantial cardiac dysfunction in patients with perforin-positive cardiac cell infiltration. METHODS AND RESULTS: Between 2003 and 2013, 2389 consecutive patients with clinically suspected CMi who underwent endomyocardial biopsies were enrolled. Endomyocardial biopsies were performed at first admission after exclusion of ischemic or valvular heart disease, and CMi was confirmed in 1717 patients. Follow-up was up to 10.1 years (median 0.47 years; interquartile range, 0.03-2.56 years) and information on vital status was obtained from official resident data files. Multivariable statistical analysis was conducted for all patients with CMi regarding significant predictors of all-cause mortality or need for heart transplantation. Multiple Cox regression analysis revealed perforin above the calculated cutoff point of 2.9 cells/mm² as a strong predictor of impaired survival with a hazard ratio of 1.881 (95% confidence interval, 1.177-3.008; P=0.008), independent of left ventricular function and other myocardial inflammation markers (CD3, macrophage-1 antigen, leukocyte function-associated antigen-1, human leukocyte antigen-1, and intercellular cell adhesion molecule-1). Unexpectedly, male sex emerged as another strong adverse predictor of survival in CMi (hazard ratio, 1.863; confidence interval, 1.096-3.168 [P=0.022]). Whereas left ventricular ejection fraction course is adversely affected by myocardial perforin, multivariate analysis indicates that left ventricular ejection fraction explains only part of the observed overall mortality. CONCLUSIONS: High perforin-positive cardiac cell infiltration and male sex are independent adverse predictors of long-term mortality in CMi. Furthermore, exact quantification of immunohistochemically detected infiltrates is necessary to assess the prognosis.

Asymptomatic CMV infections in long-term renal transplant recipients are associated with the loss of FcRγ from LIR-1+ NK cells.

While it is established that cytomegalovirus (CMV) disease affects NK-cell profiles, the functional consequences of asymptomatic CMV replication are unclear. Here, we characterize NK cells in clinically stable renal transplant recipients (RTRs; n = 48) >2 years after transplantation. RTRs and age-matched controls (n = 32) were stratified by their CMV serostatus and the presence of measurable CMV DNA. CMV antibody or CMV DNA influenced expression of NKG2C, LIR-1, NKp30, NKp46, and FcRγ, a signaling adaptor molecule, on CD56dim NK cells. Phenotypic changes ascribed to CMV were clearer in RTRs than in control subjects and affected NK-cell function as assessed by TNF-α and CD107a expression. The most active NK cells were FcRγ- LIR-1+ NKG2C- and displayed high antibody-dependent cell cytotoxicity responses in the presence of immobilized CMV glycoprotein B reactive antibody. However, perforin levels in supernatants from RTRs with active CMV replication were low. Overall we demonstrate that CMV can be reactivated in symptom-free renal transplant recipients, affecting the phenotypic, and functional profiles of NK cells. Continuous exposure to CMV may maintain and expand NK cells that lack FcRγ but express LIR-1.

American tegumentary leishmaniasis: T-cell differentiation profile of cutaneous and mucosal forms-co-infection with Trypanosoma cruzi.

American tegumentary leishmaniasis displays two main clinical forms: cutaneous (CL) and mucosal (ML). ML is more resistant to treatment and displays a more severe and longer evolution. Since both forms are caused by the same Leishmania species, the immunological response of the host may be an important factor determining the evolution of the disease. Herein, we analyzed the differentiation and memory profile of peripheral CD4(+) and CD8(+) T lymphocytes of patients with CL and ML and their Leishmania-T. cruzi co-infected counterparts. We measured the expression of CD27, CD28, CD45RO, CD127, PD-1 and CD57, together with interferon-γ and perforin. A highly differentiated phenotype was reflected on both T subsets in ML and preferentially on CD8(+) T cells in CL. A positive trend toward a higher T differentiation profile was found in T. cruzi-infected CL and ML patients as compared with Leishmania single infections. Association between CD8(+) T-cell differentiation and illness duration was found within the first year of infection, with progressive increase of highly differentiated markers over time. Follow-up of patients with good response to therapy showed predominance of early differentiated CD8(+) T cells and decrease of highly differentiated cells, while patients with frequent relapses presented the opposite pattern. CD8(+) T cells showed the most striking changes in their phenotype during leishmaniasis. Patients with long-term infections showed the highest differentiated degree implying a relation between T differentiation and parasite persistence. Distinct patterns of CD8(+) T differentiation during follow-up of different clinical outcomes suggest the usefulness of this analysis in the characterization of Leishmania-infected patients.

A Challenging Cutaneous T-Cell Lymphoma.

Cutaneous peripheral T-cell lymphomas not otherwise specified (CPTL-NOS) are rare neoplasms accounting for just 2% of cutaneous peripheral T-cell lymphomas (CPTL). Only very few case series have been reported. They represent a phenotypically and prognostically heterogenous group of CPTL that do not fit into any of CPTL well-defined subtypes. The authors report a case of a 64-year-old man with simultaneous plaque-like lesions and disseminated nodules growing rapidly on the face, trunk, and extremities over a 6-month period. There was no a history of preceding patches, erythematous plaques, rash, or pruritic lesions. These lesions were extending over 80% of the skin surface. Histopathologic analysis revealed dense diffuse infiltrates composed of mostly medium-sized to large lymphoid cells throughout the entire dermis without epidermotropism. Neoplastic cells were atypical with markedly pleomorphic nuclei. Immunohistochemistry showed that the tumor cells were positive for CD3, CD4, and CD5 with a loss of CD7. They were negative for CD20, CD8, CD56, CXCL13, PD1, TIA-1, granzyme-B, perforin, CD25, and CD30. The proliferative fraction was low, with MIB-1 labeling less than 10% of cells. The authors diagnosed the patient with primary CPTL-NOS. Despite the rarity of these tumors, clinicians as well as dermatopathologists and pathologists should be familiar with these rare CPTL especially because most of these lymphomas have an aggressive behavior and exhibit an unfavorable prognosis.

Primary Cutaneous T-cell Lymphoma With Coexpression of T-Cell Receptors αß and γδ.

T lymphocytes belong to 2 distinct sublineages that express either αß or γδ T-cell receptor (TCR) complex. Although malignancy is a great instigator of lineage infidelity, as exemplified by aberrant expression of numerous lineage markers in lymphoma cells, malignant T cells rarely coexpress αß and γδ TCR complexes. Similarly, only rare cases of CD4/CD8 double-positive primary cutaneous T-cell lymphoma have been reported. In this report, we describe a remarkable case of primary cutaneous T-cell lymphoma coexpressing αß and γδ TCR complexes, strong diffuse CD8, and a very restricted coexpression of CD4 and CD8. A 66-year-old man was referred to our center for treatment of a persistent eczematoid eruption of 6 years of duration. An initial biopsy demonstrated not only marked spongiosis, but also an epidermotropic population of CD4 small mature T cells with partial expression of CD8. The process remained indolent for another year, followed by an abrupt progression with development of plaques and tumors. Repeat biopsies of these lesions demonstrated a superimposed population of large anaplastic T cells extensively involving the dermis and epidermis. The large cells showed a strong uniform expression of CD3, CD8, CD45RA, CD5, granzyme, TIA1, perforin, TCR-ß, and TCR-γ and a weaker but unambiguous expression of CD4, CD25, CD2, and CD56. TCR gene rearrangement studies showed clonal rearrangements for TCR-ß and TCR-γ with identical peaks to those seen in the biopsy from a year earlier. The patient developed lymphadenopathy, with a biopsy showing nodal involvement by a morphologically and phenotypically identical neoplastic T-cell population. The disease showed partial response to systemic chemotherapy with development of new plaques, but these new lesions have regressed with radiation therapy.

Impaired T-bet-pSTAT1α and perforin-mediated immune responses in the tumoral region of lung adenocarcinoma.

BACKGROUND: In spite of modern therapies for non-small-cell lung cancer (NSCLC), prognosis for many patients is still poor and survival rates are low. Immunotherapy is the possibility to improve the lung immune response surrounding the tumour. However, this approach requires detailed understanding of the local immune-responses of NSCLC patients. METHODS: We analysed samples from three different regions within the lungs of NSCLC patients, whereas we distinguished between patients suffering from adenocarcinoma and squamous cell carcinoma. Expression of type 1 T helper (Th1)/type 1 cytotoxic (Tc1) factors was assessed by quantitative real-time PCR, western blot analyses or immunohistochemistry. Cytotoxic cell activity of CD8(+) T cells was determined via co-culture with autologous tumour cells and apoptosis assay. RESULTS: We found decreased levels of the transcription factor T-box expressed in T cells (T-bet or Tbx21) and of the downstream activated IFN-γ-dependent pSTAT1α isoform in the lung tumour areas of patients with NSCLC as compared with tumour-free control regions. In these patients, reduced T-bet and pSTAT1α levels were found associated with increased immunosuppressive markers like cytotoxic T lymphocyte-associated protein 4, programmed cell death 1 and with a suppression of the Th1 cell cytokine production and Tc1 cell activity. CONCLUSIONS: These findings confirm a central role of T-bet in targeted immunotherapy for patients with NSCLC.

Cytotoxic Molecule-positive Epstein-Barr Virus-associated Peripheral T-cell Lymphoma in a 20-Month-old Child: A Case Report and Review of the Literature.

Peripheral T-cell lymphoma (PTCL) is rare in children. Expression of cytotoxic molecules (CM) in nodal PTCL has unique clinicopathologic features, including an Epstein-Barr virus (EBV) association. However, CM+, EBV-associated PTCL is extremely rare in the childhood, with only 1 study having been reported to date, including both pediatric and adult patients. We report a case of CM+ PTCL in a 20-month-old boy with left neck lymphadenopathy as well as multiple visceral lesions. A biopsied lymph node was diffusely infiltrated by atypical lymphoid cells with a CD4/CD8, granzyme B+, perforin+, and TIA-1+ phenotype, and EBV positivity by in situ hybridization. Rearrangements of the TCR γ-chain and ß-chain genes were demonstrated by polymerase chain reaction. Ancillary genetic studies detected trisomy 2, trisomy 10, a structurally abnormal 6p, and additional copies of the IRF4 gene. Multiple bone marrow biopsies failed to show any evidence of tumor, histiocytic hyperplasia, or hemophagocytosis. This lesion was therefore diagnosed as "CM+, EBV-associated high-grade peripheral T-cell lymphoma." After 5 cycles of chemotherapy, the patient was in remission 8 months following initial diagnosis. To our knowledge, this represents the youngest child with this rare tumor in the published literature, and showing an unusually favorable initial response to therapy.

A Meta-analysis of the Significance of Granzyme B and Perforin in Noninvasive Diagnosis of Acute Rejection After Kidney Transplantation.

BACKGROUND: Previous studies have reported that granzyme B (GZMB) and perforin (PRF) could serve as noninvasive biomarkers in the diagnosis of acute rejection (AR) after kidney transplant. Yet, their noninvasive diagnostic value in clinical practice is still unknown. METHODS: To assess the noninvasive diagnostic performance of GZMB and PRF for AR, we performed a systematic search. After reviewing published studies in which both GZMB and PRF were detected, data on the diagnostic accuracy of separate and combined evaluation of GZMB and PRF were pooled. RESULTS: Across 16 studies (680 subjects), summary sensitivity, specificity, positive likelihood ratios, and negative likelihood ratios with 95% confidence intervals were calculated. For overall GZMB analysis, the indices were 0.76 (0.71-0.81), 0.86 (0.82-0.89), 4.58 (3.36-6.25), and 0.32 (0.22-0.47), respectively. For overall PRF analysis, the indices were 0.83 (0.78-0.88), 0.86 (0.82-0.89), 4.82 (3.66-6.35), and 0.26 (0.18-0.37), respectively. Subgroup analyses showed similar results compared to overall study analyses. In analyses of combined evaluation of GZMB and PRF, the above indices were 0.65 (0.53-0.76), 0.96 (0.91-0.98), 12.66 (5.83-27.50), and 0.40 (0.23-0.69), respectively, when both markers were positive. The probability of developing AR in kidney transplant recipients increased from 15% to 73% when both GZMB and PRF tests were positive and was reduced to 2% if that were negative. CONCLUSIONS: Currently, neither GZMB nor PRF, if evaluated alone, could be a convincing noninvasive diagnostic marker for AR in clinical practice. Combined use of PRF and GZMB post-kidney transplant may be a better choice in AR evaluation to direct allograft biopsy execution and earlier therapeutic intervention.

Expression and Localization of Granzymes and Perforin in Human Calcific Aortic Valve Disease.

BACKGROUND AND AIM OF THE STUDY: Calcified aortic valve disease (CAVD) is an actively regulated disease that shares pathophysiological hallmarks with atherosclerosis. One of these common features is extracellular matrix (ECM) remodeling, which consists of a dynamic degradation and deposition of the ECM composition. Granzymes (Grs) are ECM- degrading and pro-apoptotic proteases that have been detected in atherosclerotic lesions, but their role in CAVD remains unknown. METHODS: The expression of granzymes and perforin was characterized in heavily stenotic valves (n = 20) and control valves (n = 6) using quantitative RT-PCR and immunohistochemistry. RESULTS: Quantitative RT-PCR revealed that levels of granzymes A, B, H, K and M mRNA were 4.9-fold (p < 0.001), 7.1-fold (p < 0.001), 4.6-fold (p < 0.001), 4.7-fold (p < 0.001) and 2.8-fold (p = 0.069) higher, respectively, in stenotic aortic valves than in control valves. Perforin mRNA levels were 3.6-fold (p < 0.001) higher in stenotic valves than in control valves. Granzyme A immunohistochemical positivity was observed in mast cells and lymphocytes, granzyme H in mast cells but not in lymphocytes, and granzyme K in lymphocytes but not in mast cells. A statistical analysis was also performed to investigate the effect of statin treatment on granzyme expression, but no differences were found when compared to non-statin-treated patients. CONCLUSIONS: The data acquired showed that CAVD is characterized by an increased expression of granzymes A, B, H, K, and perforin.

Therapeutic immunisation plus cytokine and hormone therapy improves CD4 T-cell counts, restores anti-HIV-1 responses and reduces immune activation in treated chronic HIV-1 infection.

BACKGROUND: This randomised, open label, phase I, immunotherapeutic study investigated the effects of interleukin (IL)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant human growth hormone (rhGH), and therapeutic immunisation (a Clade B DNA vaccine) on combination antiretroviral therapy (cART)-treated HIV-1-infected individuals, with the objective to reverse residual T-cell dysfunction. METHODS: Twelve HIV-1(+) patients on suppressive cART with baseline CD4 T-cell counts >400 cells/mm(3) blood were randomised into one of three groups: (1) vaccine, IL-2, GM-CSF and rhGH (n=3); (2) vaccine alone (n=4); or (3) IL-2, GM-CSF and rhGH (n=5). Samples were collected at weeks 0, 1, 2, 4, 6, 8, 12, 16, 24 and 48. Interferon (IFN)-γ, IL-2, IL-4 and perforin ELISpot assays performed at each time point quantified functional responses to Gag p17/p24, Nef, Rev, and Tat peptides; and detailed T-cell immunophenotyping was undertaken by flow cytometry. Proviral DNA was also measured. RESULTS: Median baseline CD4 T-cell count was 757 cells/mm(3) (interquartile range [IQR] 567-886 cells/mm(3)), median age 48 years (IQR 42-51 years), and plasma HIV-1-RNA <50 copies/ml for all subjects. Patients who received vaccine plus IL-2, GM-CSF and rhGH (group 1) showed the most marked changes. Assessing mean changes from baseline to week 48 revealed significantly elevated numbers of CD4 T cells (p=0.0083) and improved CD4/CD8 T-cell ratios (p=0.0033). This was accompanied by a significant reduction in expression of CD38 on CD4 T cells (p=0.0194), significantly increased IFN-γ and IL-2 production in response to Gag (p=0.0122) and elevated IFN-γ production in response to Tat (p=0.041) at week 48 compared to baseline. Subjects in all treatment groups showed significantly reduced PD-1 expression at week 48 compared to baseline, with some reductions in proviral DNA. CONCLUSIONS: Multifarious immunotherapeutic approaches in the context of fully suppressive cART further reduce immune activation, and improve both CD4 T-lymphocyte counts and HIV-1-specific T-cell responses (NCT01130376).

Immunohistochemical expression of perforin in lichen planus lesions.

BACKGROUND: Lichen planus (LP) is a chronic inflammatory papulosquamous skin disease characterized by epidermal basal cell damage and a particular band-like infiltrate predominantly of T cells in the upper dermis. It is characterized by the formation of colloid bodies representing apoptotic keratinocytes. The apoptotic process mediated by CD8+ cytotoxic T lymphocytes and natural killer cells mainly involves two distinct pathways: the perforin/granzyme pathway and the Fas/FasL pathway. So far, little is known regarding the role of perforin-mediated apoptosis in LP. AIM: Is to study the expression and distribution of perforin in the epidermis and dermis of lesional LP skin. MATERIALS AND METHODS: Skin biopsy specimens from lesional skin of 31 patients with LP and 10 healthy persons were analyzed by immunohistochemistry. RESULTS: Significant accumulation of perforin + cells was found in both epidermis and dermis of LP lesions compared with healthy skin. Perforin expression was significantly upregulated in the epidermis of LP lesions. CONCLUSION: Accumulation of perforin + cells in the epidermis of LP lesions suggest a potential role of perforin in the apoptosis of basal keratinocytes.

Partial Activation of natural killer and γδ T cells by classical swine fever viruses is associated with type I interferon elicited from plasmacytoid dendritic cells.

Vaccination with live attenuated classical swine fever virus (CSFV) vaccines can rapidly confer protection in the absence of neutralizing antibodies. With an aim of providing information on the cellular mechanisms that may mediate this protection, we explored the interaction of porcine natural killer (NK) cells and γδ T cells with CSFV. Both NK and γδ T cells were refractory to infection with attenuated or virulent CSFV, and no stimulatory effects, as assessed by the expression of major histocompatibility complex (MHC) class II (MHC-II), perforin, and gamma interferon (IFN-γ), were observed when the cells were cultured in the presence of CSFV. Coculture with CSFV and myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs) showed that pDCs led to a partial activation of both NK and γδ T cells, with upregulation of MHC-II being observed. An analysis of cytokine expression by infected DC subsets suggested that this effect was due to IFN-α secreted by infected pDCs. These results were supported by ex vivo analyses of NK and γδ T cells in the tonsils and retropharyngeal lymph nodes from pigs that had been vaccinated with live attenuated CSFV and/or virulent CSFV. At 5 days postchallenge, there was evidence of significant upregulation of MHC-II but not perforin on NK and γδ T cells, which was observed only following a challenge of the unvaccinated pigs and correlated with increased CSFV replication and IFN-α expression in both the tonsils and serum. Together, these data suggest that it is unlikely that NK or γδ T cells contribute to the cellular effector mechanisms induced by live attenuated CSFV.

Presence of perforin in endomyocardial biopsies of patients with inflammatory cardiomyopathy predicts poor outcome.

AIMS: Intramyocardial inflammation is considered an adverse prognostic factor in inflammatory cardiomyopathy (CMi). However, the precise nature of immune system factors relevant for the prediction of long-term course remains elusive. The aim of this study was to analyse the prognostic relevance of perforin in a large cohort of patients with CMi. METHODS AND RESULTS: We investigated 495 consecutive patients with suspected CMi, undergoing endomyocardial biopsies (EMBs), and examined haemodynamic measurements after a long follow-up period (interquartile range 10.2-37.1 months). In EMBs, myocardial inflammation was assessed by histology and immunohistology. At follow-up, 388 patients (Group I) showed stable mild dysfunction or significant improvement, with LVEF rising from 46.2 ± 14.8% to 64.3 ± 12.3% (P < 0.0001). Lack of improvement of LV function or significant deterioration of LVEF from 42.1 ± 14.2% to 32.3 ± 11.6% (P < 0.0001) was observed in 107 patients (Group II). Multivariable statistical analysis of LVEF and immunohistochemical parameters in all patients revealed that the single most important predictor of LVEF development was detection of perforin in EMBs, with an odds ratio (OR) of 7.922 [95% confidence interval (CI) 4.380-14.326; P < 0.001] for deteriorating LVEF. Importantly, baseline LVEF (OR 0.962), LV end-diastolic diameter (OR 1.847), and other immmunohistochemical parameters (CD3, Mac-1, CD45R0, LFA-1, HLA-1, and ICAM-1) made minor or insignificant contributions to LVEF course in these 495 patients. CONCLUSIONS: In this EMB-based analysis of the long-term course of CMi we identified, for the first time, that detection of perforin in the myocardium is a key predictor of LVEF course.