Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing.

BACKGROUND: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. METHODS: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. FINDINGS: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. INTERPRETATION: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. FUNDING: Foundation for Liver Research, London.

Exposure to Contaminated River Water is Associated with Early Hatching and Dysregulation of Gene Expression in Early Life Stages of the Endangered Copper Redhorse (Moxostoma hubbsi).

The copper redhorse (Moxostoma hubbsi) is an endangered fish that spawns exclusively in the Richelieu River (Quebec, Canada). Tributaries of the Richelieu are contaminated with high levels of current-use pesticides, which may impact early-life stage (ELS) copper redhorse and other native fishes. We assessed the effects of exposure to contaminated river water on ELS copper redhorse and river redhorse (Moxostoma carinatum), a related fish that shares the copper redhorse's spawning grounds and nursery habitat. A riverside flow-through system was used to expose copper and river redhorse embryos (1000 each) to Richelieu River water or laboratory water as a control. Fish were maintained until 14 days posthatch, and water samples were taken daily for chemical analysis. Following a heavy rain event, concentrations of two neonicotinoid pesticides, clothianidin and thiamethoxam, exceeded water quality guidelines for aquatic life (20 ng/L). Using nontargeted screening, we tentatively identified an additional 24 pharmaceutical and personal care products and 23 pesticides in river water. Effects of river water on ELS fish were observed in both species, but the copper redhorse appeared to be more sensitive. Fish exposed to river water hatched 10.7 (copper redhorse) and 2.4 (river redhorse) cumulative degree days earlier than controls. Copper redhorse survival was significantly lower in river water (73 ± 16%) compared to laboratory water (93 ± 3%), whereas river redhorse survival was similar between treatments (84 ± 6% and 89 ± 4%, respectively). Sequencing of copper redhorse larvae RNA revealed 18 differentially expressed genes (DEGs) following 14 days of exposure to river water. Eight up-regulated DEGs were linked to immune function and injury response, and seven down-regulated DEGs were involved with digestion and nutrient absorption. The present study provided valuable data on the effects of ELS exposure to a real-world mixture of contaminants in two fish species of concern. Environ Toxicol Chem 2022;41:1950-1966. © 2022 SETAC.

Has the non-resection rate decreased during the last two decades among patients undergoing surgical exploration for pancreatic adenocarcinoma?

PURPOSE: To determine if improvement in imaging reduces the non-resection rate (NRR) among patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: From 2000 to 2019, 751 consecutive patients with PDAC were considered eligible for a intention-to-treat pancreatectomy and entered the operating room. In April 2011, our institution acquired a dual energy spectral computed tomography (CT) scanner and liver diffusion weighted magnetic resonance imaging (DW-MRI) was included in the imaging workup. We consequently considered 2 periods of inclusion: period #1 (February 2000-March 2011) and period #2 (April 2011-August 2019). RESULTS: All patients underwent a preoperative CT scan with a median delay to surgery of 18 days. Liver DW-MRI was performed among 407 patients (54%). Median delay between CT and surgery decreased (21 days to 16 days, P < .01), and liver DW-MRI was significantly most prescribed during period #2 (14% vs 75%, P < .01). According to the intraoperative findings, the overall NRR was 24.5%, and remained stable over the two periods (25% vs 24%, respectively). While vascular invasion, liver metastasis, and carcinomatosis rates remained stable, para-aortic lymph nodes invasion rate (0.4% vs 4.6%; P < 0.001) significantly increased over the 2 periods. The mean size of the bigger extra pancreatic tumor significantly decrease (7.9 mm vs 6.4 mm (P < .01), respectively) when the resection was not done. In multivariate analysis, CA 19-9 < 500 U/mL (P < .01), and liver DW-MRI prescription (P < .01) favoured the resection. CONCLUSIONS: Due to changes in our therapeutic strategies, the NRR did not decrease during two decades despite imaging improvement.

Patient outcome according to the 2017 international consensus on the definition of borderline resectable pancreatic ductal adenocarcinoma.

BACKGROUND/OBJECTIVE: We evaluated the usefulness of the 2017 definition of borderline pancreatic ductal adenocarcinoma (BR-PDAC) in fit patients (performance status 0-1) based on anatomical (A) and biological dimensions (B). METHODS: From 2011 to 2018, 139 resected patients with BR-PDAC according to the 2017 definition were included: 18 patients underwent upfront pancreatectomy (CA 19-9 > 500 U/mL and/or regional lymph node metastasis; BR-B group), and 121 received FOLFIRINOX (FX) induction chemotherapy and were divided into BR-A (CA 19-9 < 500 U/mL, no regional lymph node metastasis; n = 68) and BR-AB (CA 19-9 > 500 U/mL and/or regional lymph node metastasis; n = 53) groups. RESULTS: The 3 groups were comparable according to patient characteristics (except for back pain (P < .01) and CA 19-9 (P < .01)), intraoperative data, and postoperative courses. BR-AB patients required more venous resections (P < .01). The 3 groups were comparable on pathologic findings, except that BR-B patients had more lymph node invasions (P = .02). Median overall survival (OS) of the 121 patients was 45 months. In multivariate analysis, venous resection (P = .039) and R1 resection (P = .012) were poorly linked with OS, whereas BR-A classification (P < .01) independently favored OS. Median survival times of BR-A, BR-AB, and BR-B groups were undetermined, 27 months, and 20 months (P < .001), respectively. CONCLUSIONS: The 2017 definition was relevant for sub-classifying patients with BR-PDAC. The anatomical dimension (BR-A) was a favorable prognostic factor, whereas the biological dimension (BR-AB and BR-B) poorly impacted survival.

Targeting senescent cells alleviates obesity-induced metabolic dysfunction.

Adipose tissue inflammation and dysfunction are associated with obesity-related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug-inducible "suicide" genes driven by the p16Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra-abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity-related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity-related metabolic dysfunction and its complications.

Outcomes of pancreatic adenocarcinoma that was not resected because of isolated para-aortic lymph node involvement.

PURPOSE: Survival appears to be poor in cases of pancreatic ductal adenocarcinoma (PDAC) with para-aortic lymph node involvement (PALN+). However, resection is still performed in these cases because the prognostic impact of PALN+remains controversial. METHODS: PALN+was intraoperatively found in 14 patients (4.8%) with resectable PDAC who consequently did not undergo pancreatectomy. RESULTS: The median overall survival time after laparotomy was 21 months. The 1- and 3-year overall survival rates were 58.3% and 25%, respectively. CONCLUSIONS: We support the advisability of reconsidering pancreatectomy in patients with intraoperatively detected PALN+because the reported survival of such patients who undergo pancreatectomy is poorer than the survival observed for patients in our series.

Resectable pancreatic head adenocarcinoma: Is R0 resection an illusion? Genetic evaluation of venous resection margin affirmed unrecognized disease.

PURPOSE: To assess the K-ras gene mutation in the histologically negative venous margin of a pancreaticoduodenectomy (PD) specimen and its impact on survival. METHOD: From 2007 to 2010, 22 patients underwent R0 PD for resecable pancreatic adenocarcinoma. All specimens were stained and the portal vein (PV) bed was identified by blue ink; a 2mm3 sample (including the blue ink) was cut from a microscopic free-tumor block. DNA was extracted and assessed by quantitative real time polymerase chain reaction to detect the K-ras gene mutation. Twelve specimens (55%) (kras+ group) were identified with a K-ras mutation in the venous margin resection, and 10 specimens (kras- group) did not have K-ras mutation detected in the venous margin resection. RESULTS: The two groups were comparable. Overall 3years survival of patients of kras+ group versus patients of kras- group was 0 and 17% (P=0.03), respectively. Median survival time of patients of kras+ group versus patients of kras- group was 16months vs 25months (P=0.04; 95% confidence interval [1,11-1,88]), respectively. CONCLUSION: Genetic evaluation of venous resection margin affirmed unrecognized disease with strong impact on survival in more than 50% of patients with histologically R0 resection.

New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and A1155463.

Senescent cells accumulate with aging and at sites of pathology in multiple chronic diseases. Senolytics are drugs that selectively promote apoptosis of senescent cells by temporarily disabling the pro-survival pathways that enable senescent cells to resist the pro-apoptotic, pro-inflammatory factors that they themselves secrete. Reducing senescent cell burden by genetic approaches or by administering senolytics delays or alleviates multiple age- and disease-related adverse phenotypes in preclinical models. Reported senolytics include dasatinib, quercetin, navitoclax (ABT263), and piperlongumine. Here we report that fisetin, a naturally-occurring flavone with low toxicity, and A1331852 and A1155463, selective BCL-XL inhibitors that may have less hematological toxicity than the less specific BCL-2 family inhibitor navitoclax, are senolytic. Fisetin selectively induces apoptosis in senescent but not proliferating human umbilical vein endothelial cells (HUVECs). It is not senolytic in senescent IMR90 cells, a human lung fibroblast strain, or primary human preadipocytes. A1331852 and A1155463 are senolytic in HUVECs and IMR90 cells, but not preadipocytes. These agents may be better candidates for eventual translation into clinical interventions than some existing senolytics, such as navitoclax, which is associated with hematological toxicity.

'Peripheric' pancreatic cysts: performance of CT scan, MRI and endoscopy according to final pathological examination.

OBJECTIVE: To assess the accuracy of pre-operative staging in patients with peripheral pancreatic cystic neoplasms (pPCNs). METHODS: From 2005 to 2011, 148 patients underwent a pancreatectomy for pPCNs. The pre-operative examination methods of computed tomography (CT), magnetic resonance imaging (MRI), endoscopic ultrasonography (EUS) were compared for their ability to predict the suggested diagnosis accurately, and the definitive diagnosis was affirmed by pathological examination. RESULTS: A mural nodule was detected in 34 patients (23%): only 1 patient (3%) had an invasive pPCN at the final histological examination. A biopsy was performed in 79 patients (53%) during EUS: in 55 patients (70%), the biopsy could not conclude a diagnosis; the biopsy provided the correct and wrong diagnosis in 19 patients (24%) and 5 patients (6%), respectively. A correct diagnosis was affirmed by CT, EUS and pancreatic MRI in 60 (41%), 103 (74%) and 80 (86%) patients (when comparing EUS and MRI; P = 0.03), respectively. The positive predictive values (PPVs) of CT, EUS and MRI were 70%, 75% and 87%, respectively. CONCLUSIONS: Pancreatic MRI appears to be the most appropriate examination to diagnose pPCNs accurately. EUS alone had a poor PPV. Mural nodules in a PCN should not be considered an indisputable sign of pPCN invasiveness.

Pancreatic neuroendocrine tumor: A multivariate analysis of factors influencing survival.

BACKGROUND: The outcomes of pancreatic neuroendocrine tumors are extremely diverse, and determining the best strategy, optimal timing of therapy and the therapeutic results depend on understanding prognostic factors. We determined the clinical, radiological and histological factors associated with survival and tumor recurrence for patients with pancreatic neuroendocrine tumor. METHODS: From January 1, 1991 to December 31, 2011, 127 patients with pancreatic neuroendocrine tumor underwent pancreatectomy. The variables including clinical characteristics, surgical data and pathological findings were examined by univariate and multivariate analyses. RESULTS: There were 103 patients with non-functional tumors (81%). Sixty-four patients (50%) underwent left pancreatectomy, 51 (42%) patients underwent pancreatico-duodenectomy, 12 (9%) patients underwent enucleation and 2 patients (1%) underwent central pancreatectomy. Forty-eight patients (38%) had synchronous liver metastases. Six patients (5%) required portal vein resection, and 19 (15%) patients required enlarged "en-bloc" resection of adjacent organs. The overall morbidity and mortality rates were 48% and 2.3%, respectively. The 1-, 3- and 5-year overall survival rates were 94%, 84%, and 74%, respectively. In multivariate analyses, synchronous liver metastases (p = 0.02) and portal vein resection (p < 0.01) were independent prognostic factors of survival. CONCLUSIONS: Synchronous liver metastases and portal vein resection were found to be independent factors influencing survival.

Two-stage hepatectomy: who will not jump over the second hurdle?

BACKGROUND: Two-stage hepatectomy uses compensatory liver regeneration after a first noncurative hepatectomy to enable a second curative resection in patients with bilobar colorectal liver metastasis (CLM). OBJECTIVE: To determine the predictive factors of failure of two-stage hepatectomy. METHOD: Between 2000 and 2010, 48 patients with irresectable CLM were eligible for two-stage hepatectomy. The planned strategy was a) cleaning of the left hepatic lobe (first hepatectomy), b) right portal vein embolisation and c) right hepatectomy (second hepatectomy). Six patients had occult CLM (n = 5) or extra-hepatic disease (n = 1), which was discovered during the first hepatectomy. Thus, 42 patients completed the first hepatectomy and underwent portal vein embolisation in order to receive the second hepatectomy. Eight patients did not undergo a second hepatectomy due to disease progression. RESULTS: Upon univariate analysis, two factors were identified that precluded patients from having the second hepatectomy: the combined resection of a primary tumour during the first hepatectomy (p = 0.01) and administration of chemotherapy between the two hepatectomies (p = 0.03). An independent association with impairment to perform the two-stage strategy was demonstrated by multivariate analysis for only the combined resection of the primary colorectal cancer during the first hepatectomy (p = 0.04). CONCLUSION: Due to the small number of patients and the absence of equivalent conclusions in other studies, we cannot recommend performance of an isolated colorectal resection prior to chemotherapy. However, resection of an asymptomatic primary tumour before chemotherapy should not be considered as an outdated procedure.

Immunohistochemical staining for mismatch repair proteins, and its relevance in the diagnosis of hereditary non-polyposis colorectal cancer.

BACKGROUND: Hereditary non-polyposis colorectal cancer (HNPCC) arises mostly from germline mutations of the mismatch repair genes MSH2 and MLH1. The diagnosis of HNPCC is based on a set of clinical criteria that may be too restrictive to identify all affected patients. Immunohistochemical staining (IHC) for the mismatch repair proteins, MutS homologue 2 (MSH2) and MutL homologue 1 (MLH1), reliably identifies the microsatellite instability phenotype. This study evaluated the ability of IHC to detect germline mutations in an unselected group of patients with colorectal cancer (CRC). METHODS: All patients with CRC operated on between July 2000 and March 2003, and demonstrating a loss of protein, were contacted. Following informed consent, searchs for germline mutation and methylation of the promoter were performed on normal and tumoral DNA. RESULTS: Thirty patients agreed to participate, four of whom fulfilled the Amsterdam II criteria. Loss of expression of MLH1 was found in 20 patients, and loss of expression of MSH2 in ten patients. Four of the MLH1-deficient patients had a germline MLH1 point mutation (positive predictive value (PPV) 20 (95 per cent confidence interval (c.i.) 2 to 38 per cent) and 11 had promoter methylation. Seven of the MSH2-deficient patients had a germline MSH2 point mutation (PPV 70 (95 per cent c.i. 54 to 96 per cent), and none showed promoter methylation. CONCLUSION: MLH1-deficient patients who are young or have a positive family history of cancer should be referred for genetic testing and counselling, whereas MSH2-deficient patients should be counselled in the same way as patients with HNPCC.

Novel murine B-cell lymphoma/leukemia model to study BCL2-driven oncogenesis.

The BCL-2 family has been implicated in the pathogenesis of various hematopoietic malignancies, including follicular non-Hodgkin lymphoma and B-cell chronic lymphocytic leukemia. To identify genes that act synergistically in BCL2-enforced leukemogenesis, we developed a murine B-cell lymphoma/leukemia model based on the IL-3-dependent Balb/C pro-B line (FL5.12). FL5.12 cells were stably transfected with antiapoptotic BCL-2 alone or in combination with proapoptotic BAX or nonfunctional mutant BAX, thereby creating various levels of imbalance within the BCL-2 family. Transfectants were intravenously injected into normal Balb/C mice. Whereas FL5.12 cells did not provoke leukemia, mice injected with stable transfectants died of leukemia over time. Disease incidence and latency time depended on the degree of imbalance in the BCL-2 family, supporting a model whereby BCL2 drives tumorigenesis. All mice presented with hepatosplenomegaly and leukemic FL5.12 cells in peripheral blood and bone marrow compartments. Leukemic conversion was accompanied by secondary genetic aberrations leading to clonal IL-3-responsive leukemia. Cellular transformation was independent of alterations in c-Myc or downstream apoptotic pathway. Leukemic clones retained a normal DNA damage response leading to elevated P53 and P21 levels and cell cycle arrest upon irradiation. In conclusion, our mouse model may prove a valuable tool to identify genes that cooperate in BCL2-enforced lymphoma/leukemogenesis.

Extracorporeal circulation as an additional method for cerebral protection in simultaneous carotid endarterectomy and coronary artery surgical revascularization.

Surgical management of patients with concomitant carotid and coronary artery stenosis remains controversial. Our policy was always to perform at the same time carotid endarterectomy (CE) and coronary artery bypass grafting (CABG), but it was also considered that extracorporeal circulation (ECC), because of full heparinization, hemodilution, pulsatile flow, and hypothermia could provide better cerebral protection during CE. Retrospective data of 124 patients undergoing simultaneous CE and CABGs between January 1994 and December 2001 were reviewed. CE was performed prior to ECC in 65 patients (Group 1-mean age: 70.4 years; sex ratio: 49 male/16 female) and under ECC, prior to CABGs in 59 patients (Group 2-mean age: 69.9 years; sex ratio: 46 male/13 female). Overall hospital mortality was 7.3% (9/124): cardiac-related in 5 patients, or due to septicemia (1 patient), or ARD syndrome (1 patient), or stroke in two others. Univariate analysis demonstrated overweight, unstable angina, and emergency to be significant risk factors. Bilateral carotid stenosis was a significant risk factor of neurologic event when CE was performed prior to ECC (p < 0.05). In Group 1, mortality was 9.2% (6/65), and the incidence of neurologic events was 10.7% (7/65), and was responsible for two of the early deaths in patients with bilateral carotid stenosis. In Group 2, mortality was 5.1% (3/59) but never related to CE, while the neurologic morbidity was 1.7% (1 transient ischemic attack). It is concluded that (1) hospital mortality in patients undergoing simultaneous CE and CABGs was mainly cardiac-related. (2) The combined approach of both localizations appears to be mandatory, when carotid stenosis, even asymptomatic, was hemodynamically significant, or with ulcerative lesions likely to be responsible for embolism. (3) CE, first performed under ECC, appears to be a safe procedure, combining, in terms of cerebral protection, the benefits previously called up. This approach is all the more interesting when carotid stenosis is bilateral; hypothermia < or = 28 degrees C during the carotid clamping time is obviously the optimal method for cerebral protection when ipsilateral or contralateral supply is reduced, or even absent.

Abnormal WT1 expression in the CD34-negative compartment in myelodysplastic bone marrow.

In normal bone marrow, WT1 expression is restricted to CD34+ cells. We assessed WT1 mRNA expression levels with quantitative, real-time reverse transcription polymerase chain reaction in normal, myelodysplastic (MDS) and secondary acute myeloid leukaemia (sAML) bone marrow subfractions, based on differentiation status. The highest WT1 expression was observed in the primitive CD34+ rhodamine-123 (rho) dull cells, both in healthy donors and MDS or sAML patients. In contrast to normal CD34-negative bone marrow cells, WT1 was present in CD34-negative bone marrow cells in 12 out of 13 MDS patients and two sAML samples. Further analysis of this aberrant WT1 expression was performed in the CD34-negative subfractions of three MDS patients. In one of these, WT1 expression was found exclusively in the erythroid cells. This patient was completely transfusion dependent and showed morphological dyserythropoiesis. In another MDS patient, WT1 expression was found in a non-erythroid compartment. We conclude that abnormal WT1 expression may contribute to the disturbed differentiation of haematopoietic cells in MDS patients.

Stimulation of mitogenic pathways through kinase-impaired mutants of the epidermal growth factor receptor.

Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the gamma-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [(3)H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [(3)H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [(3)H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.

Thanaka (Limonia acidissima) and deet (di-methyl benzamide) mixture as a mosquito repellent for use by Karen women.

The prevention and treatment of drug-resistant malaria is becoming increasingly difficult. On the Thai-Myanmar border multi-drug resistant strains of falciparum malaria are increasing and, because the malaria vector Anopheles bite outdoors during early evening, insecticide house-spraying or impregnated bednets provide only limited protection. Therefore, the protective efficacy of repellent formulations containing di-methyl benzamide (deet) and permethrin against local vectors was estimated, when applied to the skin, and their acceptability amongst pregnant Karen women who are at relatively high risk from malaria was assessed. Human landing catches of mosquitoes showed that almost complete protection was achieved using different formulations of 20% deet and 0.5% permethrin for up to 6 h. All-night collections from human subjects indicated that this repellent combination reduced exposure to malaria parasites by at least 65 and 85% for those transmitted by Anopheles minimus and An. maculatus, respectively, the two principal vectors in this area. Pregnant women in the camps preferred repellents which were mixed with 'thanaka', a root paste made from pulp of the wood apple tree, Limonia acidissima, used locally as a cosmetic. Apart from a temporary warming sensation where repellent thanaka was applied to the skin, the repellents were well tolerated. An intervention trial is currently in progress to determine whether deet mixed with thanaka can protect pregnant women against malaria in this part of the world. Bioassays using a laboratory strain of Aedes aegypti demonstrated that thanaka is itself slightly repellent at high dosages and the mixture with deet provides protection for over 10 h. This treatment would therefore also provide some personal protection against dengue, which is increasing locally, transmitted by Ae. aegypti and Ae. albopictus biting during the daytime.