Complete genomic characterization in patients with cancer of unknown primary origin in routine diagnostics.

BACKGROUND: In ∼3%-5% of patients with metastatic disease, tumor origin remains unknown despite modern imaging techniques and extensive pathology work-up. With long diagnostic delays and limited and ineffective therapy options, the clinical outcome of patients with cancer of unknown primary (CUP) remains poor. Large-scale genome sequencing studies have revealed that tumor types can be predicted based on distinct patterns of somatic variants and other genomic characteristics. Moreover, actionable genomic events are present in almost half of CUP patients. This study investigated the clinical value of whole genome sequencing (WGS) in terms of primary tumor identification and detection of actionable events, in the routine diagnostic work-up of CUP patients. PATIENTS AND METHODS: A WGS-based tumor type 'cancer of unknown primary prediction algorithm' (CUPPA) was developed based on previously described principles and validated on a large pan-cancer WGS database of metastatic cancer patients (>4000 samples) and 254 independent patients, respectively. We assessed the clinical value of this prediction algorithm as part of routine WGS-based diagnostic work-up for 72 CUP patients. RESULTS: CUPPA correctly predicted the primary tumor type in 78% of samples in the independent validation cohort (194/254 patients). High-confidence predictions (>95% precision) were obtained for 162/254 patients (64%). When integrated in the diagnostic work-up of CUP patients, CUPPA could identify a primary tumor type for 49/72 patients (68%). Most common diagnoses included non-small-cell lung (n = 7), gastroesophageal (n = 4), pancreatic (n = 4), and colorectal cancer (n = 3). Actionable events with matched therapy options in clinical trials were identified in 47% of patients. CONCLUSIONS: Genome-based tumor type prediction can predict cancer diagnoses with high accuracy when integrated in the routine diagnostic work-up of patients with metastatic cancer. With identification of the primary tumor type in the majority of patients and detection of actionable events, WGS is a valuable diagnostic tool for patients with CUP.

The incidence of postoperative nausea and vomiting after caesarean section in patients with hyperemesis gravidarum: a retrospective cohort study.

BACKGROUND: Postoperative nausea and vomiting is one of the most common anaesthetic complications of caesarean section. This study examined the association between hyperemesis gravidarum during pregnancy and nausea and vomiting after caesarean section. METHODS: A single-centre, retrospective cohort study, using electronic databases of patients with and without hyperemesis gravidarum, undergoing caesarean section from 2015 to 2019. The incidence and severity of postoperative nausea and vomiting were established by a review of the documentation of administration of postoperative anti-emetics within the 24-h period after surgery, and examined using univariable, multivariable binary and ordered logistic regression models. RESULTS: Data were compared for 76 patients with hyperemesis gravidarum and 315 patients without the condition. The incidence of postoperative nausea and vomiting in the hyperemesis group versus the non-hyperemesis group was 43.4% vs 29.6%, respectively. The odds of experiencing postoperative nausea and vomiting was 1.95 times higher in women with hyperemesis gravidarum than in those without (aOR 1.95, 95% CI 1.13 to 3.36, P=0.016). The odds of having more severe postoperative nausea and vomiting were greater in the hyperemesis gravidarum group (aOR 1.91, 95% CI 1.14 to 3.20, P=0.014). CONCLUSION: Patients with hyperemesis gravidarum are more likely to develop nausea and vomiting after caesarean section, and this is likely to be of greater severity than in those without the condition. This finding should assist the effective provision of intra-operative and postoperative anti-emetics for patients with hyperemesis gravidarum undergoing caesarean section.

Does EBV alter the pathogenesis of malaria?

Plasmodium falciparum infections have been implicated in immune deficiencies resulting in ineffective control of Epstein-Barr virus, thereby increasing the risk of endemic Burkitt lymphoma in children. However, the impact of Epstein-Barr virus infections on the development of immunity to P. falciparum has not been studied in depth. In this review, we examine novel findings from animal co-infection models and human immuno-epidemiologic studies to speculate on the impact of acute gammaherpesvirus co-infection on malarial disease severity. Children are often concurrently or sequentially infected with multiple pathogens, and this has implications for understanding the development of protective immunity as well as in the evaluation of vaccine efficacy.

Interleukin-32 expression is associated with a poorer prognosis in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) represent the sixth most common malignancy diagnosed worldwide. Patient's survival is low due the high frequency of tumor recurrence. Inflammation promotes carcinogenesis as well as the formation of metastasis. Indeed, proinflammatory mediators are known to stimulate the expression of specific transcription factors such as Snai1 and to increase the ability of tumor cells to migrate into distant organs. The atypical interleukin-32 (IL32) was mainly described to exacerbate inflammatory responses in rheumatoid arthritis and inflammatory bowel diseases. IL32 is expressed in various cancers but its role in HNSCC physiology is still unexplored. Here, we analyzed the expression of IL32 and its implication on HNSCC aggressiveness. We showed that patients with tumor expressing high amounts of IL32 exhibit decreased disease-free periods (20.5 mo vs. 41 mo, P = 0.0041) and overall survival (P = 0.0359) in comparison with individuals with weak IL32 tumor expression. This overexpression was negatively correlated with gender (P = 0.0292) and p53 expression (P = 0.0307). In addition, in vitro data linked IL32 expression to metastasis formation since IL32 inhibition decreased Snai1 expression and tumor cell migration in a Boyden chamber assay. Our data provide new insight into the role of IL32 in HNSCC aggressiveness.

Endoscopic diagnosis and treatment of esophageal verrucous squamous cell cancer.

Verrucous squamous cell cancer (VSCC) of the esophagus is a variant of squamous cell carcinoma. This rare entity has been described in only a handful of case reports in the literature. We sought to evaluate the endoscopic features, treatment, and outcomes related to esophageal VSCC. The medical records of all patients with esophageal VSCC seen at our institution from January 1995 to December 2010 were reviewed retrospectively. A total of 11 patients (6 men; mean age 66 years [range 57-75 years]) were identified, with a mean follow up of 4 years (range 0.5-10 years) available in nine patients after diagnosis. About half the patients smoked or consumed alcohol on a regular basis. The median time interval from onset of symptoms to diagnosis of esophageal VSCC was 2.5 years (range 1-20 years), with dysphagia being present in all patients. The majority of tumors (8 of 11) exhibited a white, warty, plaque-like appearance with superimposed Candida at endoscopy, which led solely to a diagnosis of Candida esophagitis on initial presentation. The disease was either extensive (n = 5) throughout the esophagus or localized (n = 6) often by tumor nodules or projections, with the lower third of the esophagus being most commonly involved. Initial pinch biopsies were nondiagnostic in eight (73%) of the patients. Six patients underwent esophagectomy; neoadjuvant chemoradiation therapy was provided in two. In patients treated solely with surgery and who had a preoperative endoscopic ultrasound, the latter tended to overestimate staging of the lesion relative to surgical pathologic staging. Two patients were deemed to be poor operative candidates and received only chemoradiation treatment. One patient with a T2N0 tumor by endoscopic ultrasound staging was managed symptomatically with intermittent endoscopic dilation because of significant comorbidities that precluded surgery and oncologic therapy. There has been no evidence for residual or recurrent neoplastic disease in the eight patients who received treatment with surgery and/or chemoradiation therapy. Five of six patients who underwent surgery have required intermittent endoscopic dilation of anastomotic strictures during follow up. One of the two patients who received only chemoradiation therapy has required periodic endoscopic dilation for radiation-induced esophageal stricture. Two of the nine (22%) patients have died of causes unrelated to VSCC or its treatment at last follow up. In conclusion, a high index of suspicion for esophageal VSCC should be raised by the presence of long-standing symptoms coupled with white, warty esophageal lesions seen on endoscopic evaluation. Candida overgrowth can be expected to confound the diagnosis. Despite the long duration of symptoms, surgical resection typically shows relatively low-grade tumors, consistent with the rare propensity of this variant of esophageal squamous cell carcinoma to metastasize.

Effect of a tailored behavior change program on a composite lifestyle change score: a randomized controlled trial.

To evaluate the effect of a tailored behavior change program on a composite lifestyle change score. A randomized controlled trial conducted in Belgium in 2007-08 with 314 participants allocated to a control and an intervention condition. The intervention was a tailored behavior change program (web-based and individual coaching). The dose of the coaching was chosen by the participants and registered. Outcome measures were weight, saturated fat intake, fruit and vegetable intake, physical activity, smoking status and a composite lifestyle change score. Mann-Whitney U-tests, Kruskal-Wallis tests, t-tests and one-way analyses of variance were used to compare the study conditions and three intervention dose groups (no/low, medium and high intervention dose). There were no significant differences between the study conditions or between the intervention dose groups for the individual lifestyle factors. The composite lifestyle change score was significantly higher in the high intervention dose group compared with the no/low intervention dose group (P = 0.009). The composite lifestyle change score was positively related to the intervention dose, while the individual lifestyle factors were not. Behavior change programs that target multiple lifestyle factors could be evaluated by using a composite lifestyle change score taking into account the intervention dose.

Innate lymphocyte and dendritic cell cross-talk: a key factor in the regulation of the immune response.

Dendritic cells (DC) are specialized in the presentation of antigens and the initiation of specific immune responses. They have been involved recently in supporting innate immunity by interacting with various innate lymphocytes, such as natural killer (NK), NK T or T cell receptor (TCR)-gammadelta cells. The functional links between innate lymphocytes and DC have been investigated widely and different studies demonstrated that reciprocal activations follow on from NK/DC interactions. The cross-talk between innate cells and DC which leads to innate lymphocyte activation and DC maturation was found to be multi-directional, involving not only cell-cell contacts but also soluble factors. The final outcome of these cellular interactions may have a dramatic impact on the quality and strength of the down-stream immune responses, mainly in the context of early responses to tumour cells and infectious agents. Interestingly, DC, NK and TCR-gammadelta cells also share similar functions, such as antigen uptake and presentation, as well as cytotoxic and tumoricidal activity. In addition, NK and NK T cells have the ability to kill DC. This review will focus upon the different aspects of the cross-talk between DC and innate lymphocytes and its key role in all the steps of the immune response. These cellular interactions may be particularly critical in situations where immune surveillance requires efficient early innate responses.

Regulation of vimentin by SIP1 in human epithelial breast tumor cells.

The expression of Smad interacting protein-1 (SIP1; ZEB2) and the de novo expression of vimentin are frequently involved in epithelial-to-mesenchymal transitions (EMTs) under both normal and pathological conditions. In the present study, we investigated the potential role of SIP1 in the regulation of vimentin during the EMT associated with breast tumor cell migration and invasion. Examining several breast tumor cell lines displaying various degrees of invasiveness, we found SIP1 and vimentin expression only in invasive cell lines. Also, using a model of cell migration with human mammary MCF10A cells, we showed that SIP1 is induced specifically in vimentin-positive migratory cells. Furthermore, transfection of SIP1 cDNA in MCF10A cells increased their vimentin expression both at the mRNA and protein levels and enhanced their migratory abilities in Boyden Chamber assays. Inversely, inhibition of SIP1 expression by RNAi strategies in BT-549 cells and MCF10A cells decreased vimentin expression. We also showed that SIP1 transfection did not activate the TOP-FLASH reporter system, suggesting that the beta-catenin/TCF pathway is not implicated in the regulation of vimentin by SIP1. Our results therefore implicate SIP1 in the regulation of vimentin observed in the EMT associated with breast tumor cell migration, a pathway that may contribute to the metastatic progression of breast cancer.

[Screening for uterine cervical cancer in Belgium in 2003]. / Le dépistage du cancer du col de l'utérus en Belgique: le point en 2003.

Squamous cell cancer of the uterine cervix is associated with a high morbidity and mortality worldwide and in Belgium. Its development is related to the infection by oncogenic human papillomavirus (HPV) types. Cervical cancer is preceded by dysplasic stages (squamous intraepithelial lesions) which can be detected by a Pap smear. The goal of this paper is to review the screening of uterine cervical cancer in Belgium.

Dendritic cells induce the death of human papillomavirus-transformed keratinocytes.

Although human papillomavirus (HPV) antigens are expressed in a majority of (pre)neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix, progression to invasive cancer may occur, which suggests that the presentation of viral antigens to the immune system is deficient in some SILs. To determine whether professional antigen-presenting cells die in SILs, we assayed for the apoptosis of immature dendritic cells (DC) in organotypic cultures of HPV-transformed keratinocytes, which reproduce many features of in vivo observed SILs. Unexpectedly, the infiltration of organotypic cultures by DC specifically induced the apoptosis of HPV+ tumor cells, whereas DC were not affected. In the same conditions and in coculture experiments, apoptosis was not observed in normal keratinocytes. The induction of apoptosis required membrane contacts between DC and HPV-transformed keratinocytes. Although the HPV+cell lines were sensitive to the effects of TRAIL, soluble TRAILR2-Fc did not block the DC-induced apoptosis. Furthermore, although FasL and Fas were detected on DC and HPV+ cell lines, respectively, functional analysis revealed that this pathway is not responsible for the apoptosis induced by the DC. All together these results suggest that DC may be at the interface between innate and adaptive immunity by inducing the apoptosis of (pre)neoplastic cells.

The organotypic culture of HPV-transformed keratinocytes: an effective in vitro model for the development of new immunotherapeutic approaches for mucosal (pre)neoplastic lesions.

The purpose of this study is to develop a reliable in vitro human model to test new immunotherapeutic approaches for squamous cell carcinoma that develop on mucosal surfaces. The organotypic (raft) culture permits cells to proliferate and differentiate at an air-liquid interface on a dermal equivalent support. Normal keratinocytes stratify and fully differentiate in a manner similar to the normal squamous epithelial tissues, while human papillomavirus-immortalized and established squamous carcinoma cell lines exhibit dysplastic morphologies similar to (pre)neoplastic lesions seen in vivo. We have demonstrated the ability of these organotypic cultures to be manipulated by altering the epithelial stratification with cytokines (interferon-gamma and tumor necrosis factor-alpha) and by integrating activated lymphocytes or dendritic cells into the in vitro formed epithelial sheet. This model may provide a useful tool to investigate the factors contributing to the presence and function of immunocompetent cells within a neoplastic epithelium that develops on a mucosal surface.

Regulation of NF-kappaB activity by I kappaB-related proteins in adenocarcinoma cells.

Constitutive NF-kappaB activity varies widely among cancer cell lines. In this report, we studied the expression and the role of different I kappaB inhibitors in adenocarcinoma cell lines. High constitutive NF-kappaB activity and low I kappaB-alpha expression was found in a number of these cell lines. Moreover, some of these cells showed a high p100 expression, responsible for the cytoplasmic sequestration of most of p65 complexes. Treatment of these cells with TNF-alpha or other NF-kappaB activating agents induced only weakly nuclear NF-kappaB activity without significant p100 processing and led to a very weak transcription of NF-kappaB-dependent reporter gene. Induction of NF-kappaB activity can be restored by expression of the Tax protein or by treatment with antisense p100 oligonucleotides. In MCF7 A/Z cells stably transfected with a p100 expression vector, p65 complexes were sequestered in the cytoplasm by p100. These cells showed a reduced nuclear NF-kappaB induction and NF-kappaB-dependent gene transcription following TNF-alpha stimulation. As a consequence of a competition between I kappaB-alpha and p100, cells expressing high levels of p100 respond poorly to NF-kappaB activating stimuli as TNF-alpha.

Influence of the collateral function of the circle of Willis on hemispherical perfusion during carotid occlusion as assessed by transcranial colour-coded duplex ultrasonography.

OBJECTIVES: to investigate the collateral potential of the circle of Willis with transcranial colour-coded duplex ultrasonography and common carotid artery (CCA) compression. MATERIALS AND METHODS: in 46 atherosclerotic patients without cerebrovascular disease, the functional patency of the collaterals of the circle of Willis, the anterior and posterior communicating arteries, was assessed. The Peak Systolic Velocity (PSV) decrease in the middle cerebral artery (MCA) during CCA compression between complete and incomplete circles was compared. RESULTS: in 10 (22%) patients a complete and in 36 (78%) patients an incomplete circle of Willis was found, mainly due to non-functioning posterior communicating arteries. In hemispheres with collateral supply through both the anterior and the posterior communicating artery, the median PSV decrease in the MCA during CCA compression was 43%. When the posterior, anterior or both communicating arteries (1 hemisphere) were missing the PSV decrease was 58% (p =0.003), 70% (p =0.001) and 75%, respectively. CONCLUSIONS: collateral flow from the basilar to the carotid territory is often hampered by non-functioning posterior communicating arteries. A non-functioning anterior communicating artery is rare. A complete collateral circulation provides better perfusion of the MCA during carotid occlusion as compared with collateral supply through only the anterior or the posterior communicating artery in the case of an incomplete circle of Willis.

Glutathione-dependent oxidative modification of protoporphyrin and other dicarboxylic porphyrins by mammalian and plant peroxidases.

Protoporphyrin, an intermediate in heme and chlorophyll biosynthesis, can accumulate in human and plant tissues under certain pathological conditions and is a photosensitizer used in cancer phototherapy. We previously showed that protoporphyrin and the related non-natural dicarboxylic porphyrin deuteroporphyrin are rapidly oxidized by horseradish peroxidase in the presence of some thiols, especially glutathione. This study reports that bovine lactoperoxidase, but not leucocyte myeloperoxidase, can also catalyze this reaction and that Tween and ascorbic acid are inhibitors. Exogenous hydrogen peroxide is not required and cannot replace glutathione. Deuteroporphyrin was oxidized to a unique green chlorin product with two oxygen functions added directly to the characteristic reduced pyrrole ring of the chlorin. Spectroscopic and chromatographic results suggest that protoporphyrin was oxidized not to a green chlorin, but to a much more polar red porphyrin modified by oxidative addition to the two vinyl side chains. Two related nonnatural dicarboxylic porphyrins, with ethyl or hydroxyethyl instead of vinyl side chains, are not substrates or products for this enzymatic conversion.

Thiol-dependent degradation of protoporphyrin IX by plant peroxidases.

Protoporphyrin IX (PP) is the last porphyrin intermediate in common between heme and chlorophyll biosynthesis. This pigment normally does not accumulate in plants because its highly photodynamic nature makes it toxic. While the steps leading to heme and chlorophylls are well characterized, relatively little is known of the metabolic fate of excess PP in plants. We have discovered that plant peroxidases can rapidly degrade this pigment in the presence of thiol-containing substrates such as glutathione and cysteine. This thiol-dependent degradation of PP by horseradish peroxidase consumes oxygen and is inhibited by ascorbic acid.

Generation of T lymphocytes from the epithelium and stroma of squamous pre-neoplastic lesions of the uterine cervix.

In this study, we have developed a simple and efficient technique for the isolation of viable lymphocytes from the epithelium and stroma of small pre-neoplastic squamous intraepithelial lesions (SIL) of the uterine cervix. Following the separation of the epithelium from the stroma using dispase II, both biopsy fragments were used to generate T lymphocytes. The stroma-derived lymphocytes were obtained by collecting and culturing the cells migrating out of the biopsy in the presence of IL2 (50 U/ml). An average of 0.7 x 10(6) and 1.4 x 10(6) lymphocytes could be obtained after 20 and 30 days of culture, respectively. For the expansion of lymphocytes derived from the pre-neoplastic epithelium (SIL) it was necessary to use a combination of irradiated peripheral blood mononuclear cells (PBMC) as a feeder layer with PHA (0.1%), in addition to IL2 (50 U/ml). Interestingly, these lymphocytes could be obtained using either allogeneic or syngeneic PBMCs. With this protocol, we were able to generate up to 100 x 10(6) lymphocytes from the epithelium, the majority of which were T lymphocytes.

TNF-alpha and IFN-gamma down-regulate the expression of the metastasis-associated bi-functional 37LRP/p40 gene and protein in transformed keratinocytes.

The 37 LRP/p40 molecule is a bi-functional protein in which expression is increased in a large variety of cancers in association with their metastatic phenotype. Here we present the first data concerning the 37 LRP/p40 gene promoter activity and show that it is very active in a cervix carcinoma cell line. Interestingly, despite hallmarks of a housekeeping gene, we show that the 37 LRP/p40 gene promoter can be down-regulated by two potentially anticancerous cytokines, TNF-alpha and IFN-gamma. In addition, the dual fate of the protein, i.e., being intracellularly involved in the cell translation machinery and incorporated into a 67-kDa cell surface protein functioning as a laminin receptor (67LR), is differentially affected by the treatment. Our data suggest multiple regulation levels in the control of the 67LR/37LRP/p40 molecule expression and uncover new clues for the understanding of both the control of expression of this metastasis-associated molecule and the IFN-gamma and TNF-alpha anticancerous action.

Cytokine expression in squamous intraepithelial lesions of the uterine cervix: implications for the generation of local immunosuppression.

We have addressed the notion that the progression of cancer of the uterine cervix is associated with a preferential constraint on the development of a type 1 cellular mediated response, which is necessary to efficiently eliminate (pre)neoplastic cells. Based on the importance of cytokines in the regulation of an appropriate immune response, we have evaluated the expression of IL-12p40, IL-10 and transforming growth factor-beta 1 (TGF-beta1). Using reverse transcriptase-polymerase chain reaction (RT-PCR), the expression of these three cytokines was evaluated in both low-grade (LG) and high-grade (HG) cervical squamous intraepithelial lesions (SIL) and in normal exocervix and transformation zone biopsies. Our results show that the average level of IL-12 increases within both the LG and HG SIL, compared with both control groups. Interestingly, the percentage of HG SIL expressing IL-12p40 was lower compared with LG SIL. In contrast, the expression of IL-10 increased in parallel with the severity of the lesion to a maximal level in HG SIL. Using immunohistochemistry, we ascertained the presence of IL-12 protein in SIL and IL-10 protein in the transformation zone and SIL biopsies. Both IL-12- and IL-10-producing cells were localized in the stroma, not within the SIL. Furthermore, in this study we also observed that the region of the cervix the most sensitive to lesion development, the transformation zone, was associated with higher average levels of the immunosuppressive cytokines IL-10 and TGF-beta1.

Correlation of T-helper secretory differentiation and types of antigen-presenting cells in squamous intraepithelial lesions of the uterine cervix.

This study addressed the notion that the progression of cervical cancer is associated with a T-helper 2 (TH2) immunodeviation by analysing cytokine expression in 60 cervical biopsy specimens, spanning the spectrum from normal cervical tissue to high-grade squamous intraepithelial lesions (SILs). The biopsies were analysed by immunohistochemistry for the expression of TH1 [interleukin-2 (IL2), interferon gamma (IFN gamma)] and of TH2-type cytokines (IL4, IL6). Positive cells were usually observed in the subepithelial connective tissue, where most CD4+ cells were also detected. The density of IL2+ cells was significantly lower in high-grade SILs than in normal tissues taken either from the ectocervix or from the transformation zone. In contrast, significantly higher densities of IL4+ cells and, to a lesser degree, IL6+ cells were found in SIL biopsies compared with histologically normal tissues taken from the adjacent ectocervical region. A significantly higher IL4+/CD4+ cell ratio was also found in high-grade SILs (82 per cent) than in normal cervical biopsies taken from the transformation zone of healthy women showing squamous metaplasia (27 per cent). The elevated density of TH2+ cells in SIL biopsies was associated with both the expression of HLA-DR by keratinocytes and a diminished number of intraepithelial Langerhans' cells (CD1a+). In conclusion, the increased TH2+/CD4+ cell ratio in SIL biopsies suggest the presence, during cervical carcinogenesis, of a TH2 immunodeviation that could participate in the immunoescape of preneoplastic cervical keratinocytes.

Organotypic culture of HPV-transformed keratinocytes: a model for testing lymphocyte infiltration of (pre)neoplastic lesions of the uterine cervix.

The aim of our study was to establish the relevance of an in vitro model for analysing the ability of human lymphocytes to infiltrate human papillomavirus (HPV)-associated (pre)neoplastic lesions of the uterine cervix. To mimic these lesions, we have used the organotypic raft culture of HPV-transformed keratinocytes (SiHa). The SiHa organotypic raft culture was co-cultured with resting or prestimulated (IL-2 or IL-2+anti-CD3 mAb) allogeneic peripheral blood mononuclear cells (PBMC) for 24 and 72 h. The majority of infiltrating cells were T lymphocytes. Occasional NK cells were also identified. The stimulation with IL-2+anti-CD3 mAb induced the highest number of infiltrating cells, with the maximum lymphocyte infiltration observed after 24 h of co-culture. The lymphocyte infiltration was associated with an increased number of apoptotic cells in the organotypic cultures. The ability of PBMC and purified T cell and NK cell populations to lyse HPV-transformed keratinocytes was also investigated on monolayer cultures. As expected in an allogenic model, the highest cytotoxicity was mediated by NK cells activated by IL-2 or IL-2+anti-CD3 mAb. The cytotoxic activity of T cells was weak but, interestingly, increased in the presence of phytohaemagglutinin A (PHA), assuming that T cells were able to kill HPV-infected keratinocytes when a bridge between T cells and keratinocytes was provided. In conclusion, the organotypic culture of HPV-transformed keratinocytes may provide an effective in vitro model for investigating novel T cell-based immunotherapy protocols for the treatment of HPV-associated lesions.