Hippocampus Insulin Receptors Regulate Episodic and Spatial Memory Through Excitatory/Inhibitory Balance.

It is well known that the hippocampus is a vital brain region playing a key role in both episodic and spatial memory. Insulin receptors (InsRs) are densely distributed in the hippocampus and are important for its function. However, the effects of InsRs on the function of the specific hippocampal cell types remain elusive. In this study, hippocampal InsRs knockout mice had impaired episodic and spatial memory. GABAergic neurons and glutamatergic neurons in the hippocampus are involved in the balance between excitatory and inhibitory (E/I) states and participate in the processes of episodic and spatial memory. InsRs are located mainly at excitatory neurons in the hippocampus, whereas 8.5% of InsRs are glutamic acid decarboxylase 2 (GAD2)::Ai9-positive (GABAergic) neurons. Next, we constructed a transgenic mouse system in which InsR expression was deleted from GABAergic (glutamate decarboxylase 2::InsRfl/fl, GAD2Cre::InsRfl/fl) or glutamatergic neurons (vesicular glutamate transporter 2::InsRfl/fl,Vglut2Cre::InsRfl/fl). Our results showed that in comparison to the InsRfl/fl mice, both episodic and spatial memory were lower in GAD2Cre::InsRfl/fl and Vglut2Cre::InsRfl/fl. In addition, both GAD2Cre::InsRfl/fl and Vglut2Cre::InsRfl/fl were associated with more anxiety and lower glucose tolerance. These findings reveal that hippocampal InsRs might be crucial for episodic and spatial memory through E/I balance hippocampal regulation.

Projections from the Rostral Zona Incerta to the Thalamic Paraventricular Nucleus Mediate Nociceptive Neurotransmission in Mice.

Zona incerta (ZI) is an integrative subthalamic region in nociceptive neurotransmission. Previous studies demonstrated that the rostral ZI (ZIR) is an important gamma-aminobutyric acid-ergic (GABAergic) source to the thalamic paraventricular nucleus (PVT), but whether the ZIR-PVT pathway participates in nociceptive modulation is still unclear. Therefore, our investigation utilized anatomical tracing, fiber photometry, chemogenetic, optogenetic and local pharmacological approaches to investigate the roles of the ZIRGABA+-PVT pathway in nociceptive neurotransmission in mice. We found that projections from the GABAergic neurons in ZIR to PVT were involved in nociceptive neurotransmission. Furthermore, chemogenetic and optogenetic activation of the ZIRGABA+-PVT pathway alleviates pain, whereas inhibiting the activities of the ZIRGABA+-PVT circuit induces mechanical hypersensitivity and partial heat hyperalgesia. Importantly, in vivo pharmacology combined with optogenetics revealed that the GABA-A receptor (GABAAR) is crucial for GABAergic inhibition from ZIR to PVT. Our data suggest that the ZIRGABA+-PVT pathway acts through GABAAR-expressing glutamatergic neurons in PVT mediates nociceptive neurotransmission.

The clinical characteristic of alcohol-hyperlipidemia etiologically complex type of acute pancreatitis.

OBJECTIVE: The aim of our study was to elucidate the clinical characteristics of alcoholic-hyperlipidemic etiologically complex acute pancreatitis. PATIENTS AND METHODS: We reviewed complete data from 233 patients with acute pancreatitis treated in our hospital during the period January 2017-January 2022. They were divided into three groups according to etiology: alcoholic acute pancreatitis (AAP), hyperlipidemic acute pancreatitis (HLAP), and alcoholic-hyperlipidemic acute pancreatitis (AHAP). General clinical data, co-morbidities, laboratory results, imaging data, and disease severity were analyzed and compared between groups. RESULTS: The proportion of male individuals in the AHAP group was significantly higher than that in the HLAP group (p<0.001). Age of onset was lower and the number of cases with antibiotic use was higher in the AHAP group than in the AAP group (p<0.05). Additionally, the average alcohol intake each time and weekly alcohol intake were also higher in the AHAP group than in the AAP group (p<0.05). Comparison of disease severity (moderate and severe acute pancreatitis, severe acute pancreatitis, and modified computed tomography severity index score) revealed the disease condition to be more severe in the AHAP group than in the AAP and HLAP groups (p<0.05). Accordingly, patients in the AHAP group had longer hospital stays than those in the other two groups (p<0.05). There were no significant differences in alcohol consumption, severity, or length of hospital stay in the AHAP group (p>0.05). CONCLUSIONS: The clinical characteristics of patients in the AHAP, AAP and HLAP groups were different, and the patients in the AHAP group were more likely to have a moderate to severe disease course, with longer hospital stay. As a new AP classification concept, AHAP would offer high significance for diagnosis, treatment, and prognosis.

The clinical characteristic of biliary-hyperlipidemic etiologically complex type of acute pancreatitis: a retrospective study from a tertiary center in China.

OBJECTIVE: Biliary and hyperlipidemic acute pancreatitis (AP) has become the second most common AP in China. Currently, AP is exclusively diagnosed as biliary or hyperlipidemic AP. However, as suggested by some reports, biliary and hyperlipidemic AP might coexist in a single patient. Moreover, acute lipotoxicity was shown to regulate the severity of biliary AP in the mouse model. Thus, whether these two etiologies coexist in AP patients and potentially worsen the clinical course remains unclear. To elucidate the clinical feature of a new complex type of acute pancreatitis with both biliary and hyperlipidemic etiologies. PATIENTS AND METHODS: This retrospective study included AP patients who were admitted into our department within 7 days after the onset of the disease. 267 AP patients were enrolled in this study and were classified as BAP (biliary acute pancreatitis, n=153), HLAP (hyperlipidemic acute pancreatitis, n=65) and BHAP (biliary-hyperlipidemic acute pancreatitis, n=49). All the enrolled patients met the classification criteria of biliary etiology, hyperlipidemic etiology, and both etiologies, respectively. BHAP was compared with BAP and HLAP in terms of general information, inflammatory biomarkers, organ dysfunction, disease severity and clinical outcomes. RESULTS: BHAP (41 vs. 53) patients were younger than BAP patients. Serum procalcitonin of BHAP patients was higher than BAP and HLAP patients. Serum CRP of BHAP patients was higher than BAP patients. BHAP patients had the highest diagnosis rate of severe acute pancreatitis (SAP) (46.9% vs. 17.6% or 21.5%) compared to BAP and HLAP. Prevalences of persistent respiratory, acute renal, and circulatory failure were highest in BHAP patients (44.9%, 28.6%, 12.2%, respectively). Requirements for mechanical ventilation, renal replacement therapy and vasoactive agents were also highest in BHAP patients (36.7%, 34.7%, 12.2%, respectively). Hospital stay was longer in BHAP patients (33 days) compared with BAP patients (24 days). CONCLUSIONS: Patients with both biliary and hyperlipidemic etiologies suffer from more severe clinical course of the disease and have worse prognosis than single-etiology BAP or HLAP patients in the early stage of AP (within 7 days). It should be recognized as a new etiological type named biliary-hyperlipidemic acute pancreatitis (BHAP).

Risk factors for inadvertent arterial puncture during subclavian vein catheterization.

OBJECTIVE: Central vein catheterizations facilitate the establishment of reliable venous pathways in emergent medical situations. The subclavian is an important vein for central venous catheterizations. But, inadvertent arterial punctures during subclavian vein catheterizations are more dangerous than those during jugular or femoral vein catheterizations, because of the lack of a reliable compression site. We aimed to identify risk factors for the occurrence of inadvertent arterial puncture during subclavian vein catheterizations in crowded emergency rooms. PATIENTS AND METHODS: We evaluated 190 patients undergoing bedside subclavian vein catheterizations in our emergency room, from which 62 patients experienced inadvertent arterial punctures. We evaluated possible risk factors from basic physical or laboratory tests that can easily be obtained in the ER, and performed Chi-square test, Kruskal-Wallis ANOVA, non-conditional logistic regression analysis, and receiver-operating characteristic curves to determine the cut-off values of the identified risk factors. RESULTS: We identified age, BMI, and serum pre-albumin level as significant risk factors for inadvertent arterial puncture during subclavian vein catheterization (p<0.05) through regression analyses (odds ratios of 1.043, 0.719 and 0.989; and receiver-operating characteristic curves with AUCs of 0.741, 0.818, and 0.717, respectively). The cut-off values for age, BMI and serum pre-albumin level were 66.5 years old, 21.12 and 109.5 mg/L, respectively. CONCLUSIONS: We found that patients with poor nutritional status (BMI <21.12 and serum pre-albumin <109.5 mg/L) or older than 69.5 years tended to experience more accidental arterial punctures during subclavian vein catheterizations, probably due to atrophy or diminished peri-vascular support tissues in patients with poor nutritional statuses that make it difficult to obtain adequate chest extensions.

Systematic review with meta-analysis: comparative efficacy of immunosuppressants and biologics for reducing hospitalisation and surgery in Crohn's disease and ulcerative colitis.

INTRODUCTION: Crohn's disease (CD) and ulcerative colitis (UC) have a progressive course leading to hospitalisation and surgery. The ability of existing therapies to alter disease course is not clearly defined. AIM: To investigate the comparative efficacy of currently available inflammatory bowel disease (IBD) therapies to reduce hospitalisation and surgery. METHODS: We conducted a systematic review in MEDLINE/PubMed for randomised controlled trials (RCT) published between January 1980 and May 2016 examining efficacy of biological or immunomodulator therapy in IBD. We performed direct comparisons of pooled proportions of hospitalisation and surgery. Pair-wise comparisons using a random-effects Bayesian network meta-analysis were performed to assess comparative efficacy of different treatments. RESULTS: We identified seven randomised controlled trials (5 CD; 2 UC) comparing three biologics and one immunomodulator with placebo. In CD, anti-TNF biologics significantly reduced hospitalisation [Odds ratio (OR) 0.46, 95% confidence interval (CI) 0.36-0.60] and surgery (OR 0.23, 95% CI 0.13-0.42) compared to placebo. No statistically significant reduction was noted with azathioprine or vedolizumab. Azathioprine was inferior to both infliximab and adalimumab in preventing CD-related hospitalisation (>97.5% probability). Anti-TNF biologics significantly reduced hospitalisation (OR 0.48, 95% CI 0.29-0.80) and surgery (OR 0.67, 95% CI 0.46-0.97) in UC. There were no statistically significant differences in the pair-wise comparisons between active treatments. CONCLUSIONS: In CD and UC, anti-TNF biologics are efficacious in reducing the odds of hospitalisation by half and surgery by 33-77%. Azathioprine and vedolizumab were not associated with a similar improvement, but robust conclusions may be limited due to paucity of RCTs.

Identifying multidrug resistance in previously treated tuberculosis patients: a mixed-methods study in Cambodia.

SETTING: Previously treated tuberculosis (TB) patients are a priority for drug susceptibility testing (DST) to identify cases with multidrug-resistant TB (MDR-TB). A Cambodia study found that one third of smear-positive previously treated patients had DST results. OBJECTIVE: To quantify the gaps in the detection of MDR-TB in previously treated TB patients in Cambodia, and describe health workers' perspectives on barriers, facilitators and potential interventions. DESIGN: Analysis of Cambodia's 2004-2012 case notifications and semi-structured interviews with stakeholders. RESULTS: The proportion of previously treated notifications varied significantly across provinces in 2010-2012. If there had been no attrition along the path to detecting MDR-TB among smear-positive notified cases in 2012, an estimated 75 additional MDR-TB cases could have been identified, which would double the number actually detected. Most were lost due to misclassification of previously treated patients as 'new'. Barriers include patients' reluctance to disclose and staff difficulty in eliciting treatment history, partly attributed to the availability of streptomycin (SM) only in hospitals. Facilitators include collection of sputum for DST even if previously treated patients are not receiving SM, streamlining sputum transportation and prompt reporting of results. CONCLUSION: Improved monitoring, supportive staff supervision and training, patient education, and correct classification of previously treated cases are essential for improving the detection of MDR-TB.

Increased levels of soluble vascular endothelial cadherin are associated with poor outcome in severe sepsis.

Endothelial cells are thought to play a key role in sepsis pathogenesis: vascular endothelial damage occurs in severe sepsis and multiple organ dysfunction. Soluble vascular endothelial cadherin (VE-cadherin) levels were investigated in a prospective study involving 28 consecutive critically ill patients with or without severe sepsis who were admitted to surgical intensive care; 13 healthy age-matched volunteers were included as controls. Soluble VE-cadherin levels increased significantly in patients with severe sepsis compared with ill patients without severe sepsis and healthy controls. There was a significant linear correlation between soluble VE-cadherin levels and illness severity scores. Soluble VE-cadherin levels were significantly higher in patients who died compared with survivors. In vitro cell culture showed that serum from patients with severe sepsis greatly decreased VE-cadherin staining at intercellular junctions and total VE-cadherin expression in human umbilical cord vein endothelial cells. These findings suggest that endothelial cells play an important role in the poor outcome of patients with severe sepsis.

Heterotrophic culture of Chlorella protothecoides in various nitrogen sources for lipid production.

The influences of urea, yeast extract, and nitrate as the nitrogen source on heterotrophic growth of four strains of Chlorella protothecoides were investigated in 9-day feed-batch cultures. Biomass dry weight concentration (DWC) and lipid yield (LY) of the four strains in all media were compared. The highest LY in 9 days was 654 mg/L/day by UTEX 255 in 2.4 g/L KNO(3) medium with a biomass DWC of 11.7 g/L and lipid content of 50.5%. Using green autotrophic seeds instead of yellow heterotrophic seeds improved the biomass DWC (13.1 vs. 11.7 g/L), LY (850 vs. 654 mg/L/day), and lipid to glucose consumption ratio (0.607 vs. 0.162). Moreover, 17.0 g/L DWC and 489 mg/L/day LY were obtained from the sequentially mixed-nitrogen medium, and the lipid to glucose consumption ratio was improved to 0.197 from 0.162 in 2.4 g/L nitrate medium and from 0.108 in 4.2 g/L yeast extract medium in the first batch.

[Effects of short veno-venous hemofiltration on severe acute pancreatitis].

OBJECTIVE: To estimate the effects of therapy of short veno-venous hemofiltration (SVVH, 4hr) on severe acute pancreatitis (SAP). METHODS: Twenty patients were divided randomly into hemofiltration group (HF, 10 pts) and non-hemofiltration group (NHF, 10 pts). The local, systemic manifestations were compared between two groups; and the concentration of serum pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-6, IL-8, sIL-2R) and anti-inflammatory cytokines (IL-2, IL-10) were determined at different observation points. RESULTS: In the HF and NHF group, the duration for disappearance of abdominal pain and tenderness, and amelioration for abdominal distension was (8.5 +/- 6.3) d vs. (90.0 +/- 49.0) d and (16.9 +/- 5.2) d vs. (63.4 +/- 36.0) d. CT scores at the 14th day and APACHE II scores at the 10th day was (5.6 +/- 1.3) vs. (7.7 +/- 0.8) and (4.5 +/- 1.5) vs. (7.4 +/- 2.1). The average hospital stay and cost of therapy were (33.0 +/- 20.7) d vs. (56.4 +/- 16.0) d and (53,200 +/- 16,000) vs. (89,100 +/- 25,000) RMB (P < 0.05). The concentration of 5 serum pro-inflammatory cytokines at each observation points after hemofiltration was decreased significantly (P < 0.05) in the HF group. But the concentration of the two serum anti-inflammatory cytokines was increased significantly (P < 0.05) as compared with that of the NHF group. CONCLUSIONS: Through SVVH, the imbalance of pro-inflammatory and anti-inflammatory cytokines has been corrected at early stage; thus, the severity of the disease is ameliorated significantly and the efficacy of therapy is increased.

Oral cancer risk in relation to sexual history and evidence of human papillomavirus infection.

BACKGROUND: Experimental models and analyses of human tumors suggest that oncogenic, sexually transmittable human papillomaviruses (HPVs) are etiologic factors in the development of oral squamous cell carcinoma (SCC). We conducted a population-based, case-control study to determine whether the risk of this cancer is related to HPV infection and sexual history factors. METHODS: Case subjects (n = 284) were 18-65-year-old residents of three counties in western Washington State who were newly diagnosed with oral SCC from 1990 through 1995. Control subjects (n = 477) similar in age and sex were selected from the general population. Serum samples were tested for HPV type 16 capsid antibodies. Exfoliated oral tissue collected from case and control subjects and tumor tissue from case subjects were tested for HPV DNA. Odds ratios (ORs) were calculated after adjusting for age, sex, cigarette smoking, and alcohol consumption. RESULTS: Among males only, oral SCC risk increased with self-reported decreasing age at first intercourse, increasing number of sex partners, and a history of genital warts. Approximately 26% of the tumors in case subjects contained HPV DNA; 16.5% of the tumors contained HPV type 16 DNA. The prevalence of oncogenic HPV types in exfoliated oral tissue was similar in case and control subjects. The ORs for HPV type 16 capsid seropositivity were 2.3 (95% confidence interval [CI] = 1.6-3.3) for all oral SCCs and 6.8 (95% CI = 3.0-15.2) for oral SCCs containing HPV type 16 DNA. The joint association of cigarette smoking and HPV type 16 capsid seropositivity with oral SCC (OR = 8.5; 95% CI = 5.1-14.4) was stronger than predicted from the sum of individual associations with current smoking (OR = 3.2; 95% CI = 2.0-5.2) and seropositivity (OR = 1.7; 95% CI = 1.1-2.6). CONCLUSIONS: HPV type 16 infection may contribute to the development of a small proportion of oral SCCs in this population, most likely in combination with cigarette smoking.

Examination of the role of DNA polymerase proofreading in the mutator effect of miscoding tRNAs.

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, epsilon, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered epsilon subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes epsilon. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the epsilon subunit playing a major role in the mutator effect of misreading tRNAs.

Loss of heterozygosity at 5q21-22 (adenomatous polyposis coli gene region) in oral squamous cell carcinoma is common and correlated with advanced disease.

We determined the frequency of loss of heterozygosity (LOH) at chromosome 5q21-22 (adenomatous polyposis gene region) in oral SCC from 49 patients using PCR-based assays. Of 43 informative (heterozygous) tumors, 41.9% [95% confidence interval (CI)=27.0, 57.9] contained LOH at 5q21-22. LOH at 5q21-22 was strongly associated with stage at diagnosis: 100%, (3/3), 50% (13/26), and 14% (2/14) of tumors from patients with distant metastases, regional spread, and localized disease, respectively, contained this genetic alteration (P=0.01). There were no statistically significant associations between LOH at 5q21-22 and other patient or tumor characteristics, but LOH was more commonly found in the tumors of heavy smokers, infrequent alcohol consumers, and in tumors containing either p53 mutations or HPV-DNA. In univariate analyses, LOH at 5q21-22 was associated with poor prognosis (hazard ratio=1.8, 95%, CI 0.8, 4.5); this relationship did not persist after adjustment for stage of disease (hazard ratio=1.1, 95% CI=0.4, 3.1). These data provide further evidence that inactivation of the APC gene and/or other genes at 5q21-22 is common and may be involved in the development and/or progression of oral SCC. Larger studies are needed to determine whether LOH at 5q21-22 is linked to known oral SCC etiologic factors and/or the prognosis of oral SCC patients, as well as to genetic instability at other loci involved in these malignancies.

[Pancreatic ischemia: a continuous injury factor in acute necrotic pancreatitis].

ANP model in rats was used to determine the concentration of TXB2, PGF1 alpha in plasma and the ACE activity in serum in five groups. The concentration of TXB2, PGF1 alpha in all experimental groups was significantly different from that of control group (P < 0.05). The comparison of ACE activity was just the same as the above except that of 6 h. The factors leading to pancreatic ischemia functioned continously. We conclude that pancreatic ischemia is a continuous injury factor in ANP, and there is no reperfusion-injury in the course of the disease.

Proliferation of mutators in A cell population.

A Lac- strain of Escherichia coli that reverts by the addition of a G to a G-G-G-G-G-G sequence was used to study the proliferation of mutators in a bacterial culture. Selection for the Lac+ phenotype, which is greatly stimulated in mismatch repair-deficient strains, results in an increase in the percentage of mutators in the selected population from less than 1 per 100,000 cells to 1 per 200 cells. All the mutators detected were deficient in the mismatch repair system. Mutagenesis results in a similar increase in the percentage of mutators. Mutagenesis combined with a single selection can result in a population of more than 50% mutators when a sample of several thousand cells is grown out and selected. Mutagenesis combined with two or more successive selections can generate a population that is 100% mutator. These experiments are discussed in relation to ideas that an early step in carcinogenesis is the creation of a mutator phenotype.

Chromosomal abnormalities in HPV-16-immortalized oral epithelial cells.

Human papilloma virus (HPV) type 16 has an established association with anogenital carcinoma, and to some extent with human oral squamous cell carcinoma. We hypothesize that HPV type 16 is capable of inducing chromosomal and cell cycle changes in cultured oral epithelial cells. Normal human oral epithelia] cells were immortalized with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. These cells have been in culture for more than 350 passages and over 4 years. Flow cytometry demonstrated an average of 42% nuclear aneuploidy in HPV 16-immortalized cells; 16% in normal controls (probably tetrasomy). Cytogenetic analysis demonstrated significant progression of chromosomal abnormalities. Cells at early passage (p10) showed trisomy 20, with no other major changes. At passage 18, trisomy 1q and monosomy 13 were seen in addition to trisomy 20. At passage 61 there were two distinct cell populations ('a' and 'b'), with multiple chromosomal changes including trisomy 5q,14,20 in one line and 7p,9q,llq in the other. Both populations had monosomy 3p, with monosomy 8p in one population and monosomy 13 in the other. At passage 136, the cells were essentially identical to population 'b' of passage 61. At this passage, mutation of the p53 gene was detected at codon 273 of exon 8, with G to T conversion (Arg to Leu). This was absent in the normal cells from which this line was developed. Passage 262 contained the two major cell populations, each with a sub-group with additional chromosomal changes such as 10p monosomy. Cells from passages 217 and 305 were injected into nude mice a year apart. Both failed to produce tumors, as did normal cells. In conclusion, we present an HPV type 16-immortalized oral epithelial cell line (IHGK) with extensive and progressive chromosomal abnormalities, invasive growth in culture and yet no tumor formation in nude mice. We suggest that the question as to whether HPV alone can induce transformation is still open.

Loss of the adenomatous polyposis coli gene and human papillomavirus infection in oral carcinogenesis.

Recent evidence suggests that loss of heterozygosity (LOH) of the adenomatous polyposis coli (APC) gene plays a role in colorectal tumorigenesis and other cancers. However, little is known as to whether the APC gene contributes to the pathogenesis of oral squamous cell carcinoma. To assess involvement of both the APC gene and the human papillomavirus (HPV) in the development of oral pre-malignant and malignant lesions, we analysed DNA from 14 paired oral normal and pre-malignant or malignant paraffin-embedded biopsy specimens, and DNA from cultured normal and HPV 16-immortalised oral epithelial cells for the presence of LOH of APC and for HPV infection, using PCR based techniques. LOH of APC occurred in 80% of cases of oral epithelial dysplasia, 67% of carcinoma in situ, 50% of invasive squamous cell carcinoma cases, and in the HPV 16-immortalised oral epithelial cells. HPV was detected in half of the biopsy specimens, with HPV 16 as the dominant type. More than half of the carcinoma cases were found to contain both LOH of APC and HPV infection. These results suggest that LOH of APC is an early event during oral tumorigenesis. Our findings also suggest a strong correlation between HPV infection, particularly HPV 16, and LOH of the APC gene in oral squamous cell carcinomas.

Human papilloma viruses and p53 mutations in normal pre-malignant and malignant oral epithelia.

HPV infections have been previously observed in oral cancers, and inactivation of the p53 gene has been shown to be one of the most common genetic alterations in human tumors. We examined 179 oral specimens from 70 individuals with histologic findings of either normal mucosa (n = 6) or oral disease that ranged from mild dysplasia to invasive squamous-cell carcinoma (n = 64) to determine the occurrence of both HPV infection and p53 mutations and their relationship with several clinical factors. HPV infection was detected by PCR amplification of viral DNA, and the presence of p53 mutations was assayed using the single-strand conformation polymorphism (SSCP)-PCR technique. HPV infection was found in 31% of individuals with oral disease and was not seen in healthy individuals. Mutations in exons 5, 6, 7 or 8 of the p53 gene were detected in 37.5% of patients with oral lesions and in a biopsy from 1 healthy individual who was a heavy smoker. Approximately one-third of lesions classified as pre-malignant (dysplasia and carcinoma in situ) and 42% of invasive carcinomas contained p53 mutations. The majority of these mutations were G:T transversions located within exons 7 and 8. Tumor tissues from 6 patients with oral lesions were found both to be HPV-16-positive and to contain p53 mutations; of these, 4 were poorly differentiated carcinomas that were diagnosed as late-stage disease. In this study, p53 mutations were detected in the early stages of cancer development.