Add-on Rehmannia-6-Based Chinese Medicine in Type 2 Diabetes and CKD: A Multicenter Randomized Controlled Trial.

BACKGROUND: Diabetes is the leading cause of CKD and kidney failure. We assessed the real-world effectiveness of Rehmannia-6-based Chinese medicine treatment, the most used Chinese medicine formulation, on the change in eGFR and albuminuria in patients with diabetes and CKD with severely increased albuminuria. METHODS: In this randomized, assessor-blind, standard care-controlled, parallel, multicenter trial, 148 adult patients from outpatient clinics with type 2 diabetes, an eGFR of 30-90 ml/min per 1.73 m 2 , and a urine albumin-to-creatinine ratio (UACR) of 300-5000 mg/g were randomized 1:1 to a 48-week add-on protocolized Chinese medicine treatment program (using Rehmannia-6-based formulations in the granule form taken orally) or standard care alone. Primary outcomes were the slope of change in eGFR and UACR between baseline and end point (48 weeks after randomization) in the intention-to-treat population. Secondary outcomes included safety and the change in biochemistry, biomarkers, and concomitant drug use. RESULTS: The mean age, eGFR, and UACR were 65 years, 56.7 ml/min per 1.73 m 2 , and 753 mg/g, respectively. Ninety-five percent ( n =141) of end point primary outcome measures were retrievable. For eGFR, the estimated slope of change was -2.0 (95% confidence interval [CI], -0.1 to -3.9) and -4.7 (95% CI, -2.9 to -6.5) ml/min per 1.73 m 2 in participants treated with add-on Chinese medicine or standard care alone, resulting in a 2.7 ml/min per 1.73 m 2 per year (95% CI, 0.1 to 5.3; P = 0.04) less decline with Chinese medicine. For UACR, the estimated proportion in the slope of change was 0.88 (95% CI, 0.75 to 1.02) and 0.99 (95% CI, 0.85 to 1.14) in participants treated with add-on Chinese medicine or standard care alone, respectively. The intergroup proportional difference (0.89, 11% slower increment in add-on Chinese medicine, 95% CI, 0.72 to 1.10; P = 0.28) did not reach statistical significance. Eighty-five adverse events were recorded from 50 participants (add-on Chinese medicine versus control: 22 [31%] versus 28 [36%]). CONCLUSIONS: Rehmannia-6-based Chinese medicine treatment stabilized eGFR on top of standard care alone after 48 weeks in patients with type 2 diabetes, stage 2-3 CKD, and severely increased albuminuria. CLINICAL TRIAL REGISTRY: Semi-individualized Chinese Medicine Treatment as an Adjuvant Management for Diabetic Nephropathy (SCHEMATIC), NCT02488252 .

Potential Therapeutic Targets of Rehmannia Formulations on Diabetic Nephropathy: A Comparative Network Pharmacology Analysis.

Background: Previous retrospective cohorts showed that Rehmannia-6 (R-6, Liu-wei-di-huang-wan) formulations were associated with significant kidney function preservation and mortality reduction among chronic kidney disease patients with diabetes. This study aimed to investigate the potential mechanism of action of common R-6 variations in a clinical protocol for diabetic nephropathy (DN) from a system pharmacology approach. Study Design and Methods: Disease-related genes were retrieved from GeneCards and OMIM by searching "Diabetic Nephropathy" and "Macroalbuminuria". Variations of R-6 were identified from a published existing clinical practice guideline developed from expert consensus and pilot clinical service program. The chemical compound IDs of each herb were retrieved from TCM-Mesh and PubChem. Drug targets were subsequently revealed via PharmaMapper and UniProtKB. The disease gene interactions were assessed through STRING, and disease-drug protein-protein interaction network was integrated and visualized by Cytoscape. Clusters of disease-drug protein-protein interaction were constructed by Molecular Complex Detection (MCODE) extension. Functional annotation of clusters was analyzed by DAVID and KEGG pathway enrichment. Differences among variations of R-6 were compared. Binding was verified by molecular docking with AutoDock. Results: Three hundred fifty-eight genes related to DN were identified, forming 11 clusters which corresponded to complement and coagulation cascades and signaling pathways of adipocytokine, TNF, HIF-1, and AMPK. Five variations of R-6 were analyzed. Common putative targets of the R-6 variations on DN included ACE, APOE, CCL2, CRP, EDN1, FN1, HGF, ICAM1, IL10, IL1B, IL6, INS, LEP, MMP9, PTGS2, SERPINE1, and TNF, which are related to regulation of nitric oxide biosynthesis, lipid storage, cellular response to lipopolysaccharide, inflammatory response, NF-kappa B transcription factor activity, smooth muscle cell proliferation, blood pressure, cellular response to interleukin-1, angiogenesis, cell proliferation, peptidyl-tyrosine phosphorylation, and protein kinase B signaling. TNF was identified as the seed for the most significant cluster of all R-6 variations. Targets specific to each formulation were identified. The key chemical compounds of R-6 have good binding ability to the putative protein targets. Conclusion: The mechanism of action of R-6 on DN is mostly related to the TNF signaling pathway as a core mechanism, involving amelioration of angiogenesis, fibrosis, inflammation, disease susceptibility, and oxidative stress. The putative targets identified could be validated through clinical trials.

Patients' and clinicians' expectations on integrative medicine Services for Diabetes: a focus group study.

BACKGROUND: Difference of perspective between patients and physicians over integrative medicine (IM) research and service provision remains unclear despite significant use worldwide. We observed an exceptionally low utilisation of IM and potential underreporting in diabetes. We aimed to explore the barriers and recommendations regarding service delivery and research of IM service among diabetes patients and physicians. METHODS: A 10-group, 50-participant semi-structured focus group interview series was conducted. Twenty-one patients with diverse severity of disease, comorbidities and education levels; and 29 physicians (14 conventional medicine (ConM) and 15 Chinese medicine (CM)) with diverse clinical experience, academic background and affiliation were purposively sampled from private and public clinics. Their perspectives were qualitatively analysed by constant comparative method. RESULTS: Seven subthemes regarding barriers towards IM service were identified including finance, service access, advice from medical professionals, uncertainty of service quality, uncertainty of CM effect, difficulty in understanding CM epistemology and access to medical records. Patients underreported the use of CM due to the concern over neutrality of medical advice among physicians. Inconvenience of service access, frequent follow-up, use of decoction and long-term financial burden were identified as key obstacles among patients. Regarding research design, ConM physicians emphasised standardisation and reproducibility while CM physicians emphasised personalisation. Some CM-related outcome measurements were suggested as non-communicable. Both physicians acknowledged the discordance in epistemology should be addressed by pragmatic approach. CONCLUSION: Key obstacles of CAM clinical utilisation are different between patients. Further assessment on IM should be pragmatic to balance between standardisation, reproducibility and real-world practice. Evidence-based IM programs and research should merge with existing infrastructure.

Semi-individualised Chinese medicine treatment as an adjuvant management for diabetic nephropathy: a pilot add-on, randomised, controlled, multicentre, open-label pragmatic clinical trial.

INTRODUCTION: Diabetes mellitus and diabetic nephropathy (DN) are prevalent and costly to manage. DN is the leading cause of end-stage kidney disease. Conventional therapy blocking the renin-angiotensin system has only achieved limited effect in preserving renal function. Recent observational data show that the use of Chinese medicine (CM), a major form of traditional medicine used extensively in Asia, could reduce the risk of end-stage kidney disease. However, existing clinical practice guidelines are weakly evidence-based and the effect of CM remains unclear. This trial explores the effect of an existing integrative Chinese-Western medicine protocol for the management of DN. OBJECTIVE: To optimise parameters and assess the feasibility for a subsequent phase III randomised controlled trial through preliminary evaluation on the effect of an adjuvant semi-individualised CM treatment protocol on patients with type 2 diabetes with stages 2-3 chronic kidney disease and macroalbuminuria. METHODS AND ANALYSIS: This is an assessor-blind, add-on, randomised, controlled, parallel, multicentre, open-label pilot pragmatic clinical trial. 148 patients diagnosed with DN will be recruited and randomised 1:1 to a 48-week additional semi-individualised CM treatment programme or standard medical care. Primary end points are the changes in estimated glomerular filtration rate and spot urine albumin-to-creatinine ratio between baseline and treatment end point. Secondary end points include fasting blood glucose, glycated haemoglobin, brain natriuretic peptide, fasting insulin, C peptide, fibroblast growth factor 23, urinary monocyte chemotactic protein-1, cystatin C, nephrin, transforming growth factor-ß1 and vascular endothelial growth factor. Adverse events are monitored through self-completed questionnaire and clinical visits. Outcomes will be analysed by regression models. Enrolment started in July 2015. ETHICS AND REGISTRATION: This protocol is approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (reference number UW 14-301). TRIAL REGISTRATION NUMBER: NCT02488252.

Streptococcus bovis peritonitis complicating peritoneal dialysis--a review of 10 years' experience.

OBJECTIVE: An association of Streptococcus bovis bacteremia with carcinoma of colon has been reported, but data regarding peritoneal dialysis (PD) peritonitis caused by S. bovis is scarce. In this study, we examined the clinical characteristics, associations, and outcomes of this disease entity. METHODS: The case records of patients with S. bovis PD peritonitis presenting to 2 renal centers between January 2000 and September 2010 were reviewed. Clinical features and outcomes were identified and analyzed. RESULTS: Of cultures from 23 episodes of S. bovis peritonitis in 20 patients (1.28% of all peritonitis episodes at our center), 19 (82.6%) showed S. bovis alone, and 4 (17.4%) showed mixed growth. In 7 episodes, the S. bovis was moderately resistant to penicillin G. Rates of resistance to clindamycin and erythromycin were 43.5% and 47.8% respectively. In 18 episodes (78.3%), a primary response was achieved with a first-generation cephalosporin and an aminoglycoside. In 4 episodes, a secondary response was achieved after a switch from cephalosporin to vancomycin, and in 1 episode with mixed growth, the Tenckhoff catheter had to be removed. Repeat peritonitis occurred in 3 patients at a mean of 50.0 months (range: 24.2 - 83.1 months). Of the 20 patients of S. bovis peritonitis, 10 (50%) underwent either a barium enema or a colonoscopy. One patient had history of colonic carcinoma 2 years before the peritonitis, and a subsequent work-up revealed no recurrence. Three patients had diverticulosis, and one had a concomitant sigmoid polyp. Findings in the other 6 patients were normal. No colorectal malignancy had developed in the remaining 10 patients after a mean follow-up of 76.6 months (range: 0.8 - 125.1 months). CONCLUSIONS: Outcomes in S. bovis PD peritonitis were favorable, and an association with colorectal cancer was not found in our patients. Routine colonoscopy in these patients remains controversial and should be individualized.

Clinical pictures and novel mutations of WT1-associated Denys-Drash syndrome in two Chinese children.

Denys-Drash syndrome (DDS) is characterized by early onset of nephropathy, genitalia malformation, and Wilms' tumor, where WT1 is the gene that is mutated in most patients. We report two de novo mutations in WT1 found in two Chinese DDS children. Patient 1 was a boy with complete DDS who was presented with progressive nephropathy, unilateral Wilms' tumor, bilateral cryptorchidism, and renal histology showing diffuse mesangial sclerosis (DMS). When the patient was 24 months old, a liver ultrasound showed multiple nodules, and the patient died of pneumonia 1 month later. The de novo novel mutation, c.1130A>T (p.His377Leu), was identified; the mutation replaces histidine with leucine in the zinc finger (Znf) structure and is predicted to change the local spatial structure of the protein. Patient 2 had 46 XX with incomplete DDS and presented with normal genitalia, proteinuria, unilateral Wilms' tumor with renal pedicle lymph node metastasis, and renal histology showing DMS. Her renal function remains normal after 48 months. A de novo mutation, c.1168C>T (p.Arg390Term), was identified; it truncates 60 amino acids at the C terminus, and it is predicted to result in loss of the DNA-binding capacities of the WT1 protein.

Severe hypocalcaemia and hyperphosphataemia caused by oral sodium phosphate fleet solution in a haemodialysis patient after parathyroidectomy.

We report a case with severe electrolyte disturbance after the use of oral sodium phosphate solution (OSPS). A 69-year-old patient on haemodialysis received 45 mL of OSPS for bowel preparation. He had symptomatic hypocalcaemia with a serum calcium level of 0.95 mmol/L and serum phosphate level of 4.73 mmol/L. He was treated with haemodialysis and intravenous calcium supplementation. This patient had total parathyroidectomy recently leading to the absence of parathyroid hormone response. OSPS has been reported to cause life-threatening electrolyte disturbance especially in patients with renal failure. We suggest the use of safer alternatives for bowel preparations in renal failure patients.

Colonic diverticulosis as a risk factor for peritonitis in Chinese peritoneal dialysis patients.

OBJECTIVES: The risk of development of enteric peritonitis in Chinese peritoneal dialysis (PD) patients with colonic diverticulosis is not known. There have been no reports on whether colonic diverticulosis may affect peritonitis outcomes. The objectives of this study were to examine whether colonic diverticulosis is a risk factor for the development of enteric peritonitis and to study its influence on the outcome of enteric peritonitis. PATIENTS AND METHODS: All continuous ambulatory PD patients that had barium enema and colonoscopic examinations performed between January 1994 and January 2006 were included. They were divided into 2 groups: patients with diverticulosis and patients without diverticulosis. Their demographic and clinical characteristics, colonic examination findings, and peritonitis data were compared and analyzed. RESULTS: 104 Chinese patients received 110 colonoscopies and 51 barium enema examinations. 25 patients (24.0%) had colonic diverticulosis. Patients with diverticulosis were older (65.4 +/- 14.7 vs 58.4 +/- 14.0 years, p = 0.033). The most common site of involvement of diverticulosis was the ascending colon (56%). 128 episodes of enteric peritonitis were recorded in 49 patients. Compared with patients without enteric peritonitis, more patients in the enteric peritonitis group had diverticulosis (38.8% vs 10.9%, p = 0.001) and diverticulosis most often involved the ascending colon (20.4% vs 7.3%, p = 0.082). Multivariate logistic regression analysis showed that the presence of diverticulosis (hazard ratio 5.17, 95% confidence interval 1.86 - 14.40; p = 0.002) and diverticulosis involving the ascending colon (hazard ratio 6.89, 95% confidence interval 1.43 - 33.32, p = 0.016) were independent risk factors for the development of enteric peritonitis. Enteric peritonitis in patients with diverticulosis had a higher but nonsignificant treatment failure rate (26.9% vs 18.4%, p = 0.282). CONCLUSION: In this selected cohort of PD patients with indications of colonic examinations, diverticulosis, especially involving the ascending colon, may be a risk factor for the development of enteric peritonitis. Colonic diverticulosis does not appear to affect the outcome of enteric peritonitis. Further studies are warranted to determine ways to prevent enteric peritonitis in PD patients with diverticulosis.

Mechanism of chronic aristolochic acid nephropathy: role of Smad3.

Aristolochic acid nephropathy (AAN) has become a worldwide disease and is the most severe complication related to the use of traditional Chinese medicine. However, the pathogenic mechanisms of AAN remain unclear and therapies are limited. The present study tested the hypothesis that transforming growth factor (TGF)-beta/Smad3 may be a key pathway leading to chronic AAN. This was examined in vivo in Smad3 wild-type/knockout (WT/KO) mice and in vitro in tubular epithelial cells with knockdown of Smad2 or Smad3. Results revealed that chronic administration of aristolochic acid (AA) resulted in a severe AAN characterized by progressive renal dysfunction and tubulointerstitial fibrosis including epithelial-mesenchymal transition (EMT) in Smad3 WT mice, but not in Smad3 KO mice, suggesting a critical role for Smad3 in the development of AAN. This was further tested in vitro. We found that AA was able to activate Smad signaling to mediate EMT and renal fibrosis via both TGF-beta-dependent and JNK/MAP kinase-dependent mechanisms because blockade of JNK and specific knockdown of Smad3, but not Smad2, were able to attenuate AA-stimulated collagen matrix expression and EMT. In conclusion, TGF-beta/Smad3 may be an essential mediator for chronic AAN. Results from this study indicate that specific blockade of the TGF-beta/Smad3 signaling pathway may have therapeutic potential for chronic AAN.

Bradykinin and high glucose promote renal tubular inflammation.

BACKGROUND: The role of the kallikrein-kinin system in diabetic nephropathy remains controversial. METHODS AND RESULTS: High-glucose (HG) super-induced interleukin (IL)-6, CCL-2, transforming growth factor (TGF)-beta, vascular endothelial growth factor (VEGF) and B(2)K receptor (B(2)KR) mRNA in cultured proximal tubular epithelial cells (PTEC), whereas bradykinin (BK) upregulated IL-6, CCL-2 and TGF-beta mRNA. HG activated mitogen-activated protein kinase (MAPK) p42/p44 and protein kinase C (PKC) signals, whereas BK only activated MAPK. Tubular expression of these mediators and tissue kallikrein 1 (KLK1) was confirmed in human diabetic kidney biopsies. Inhibition of MAPK p42/p44 by PD98059 partially reduced HG and BK induction of IL-6, CCL-2 and TGF-beta, whereas inhibition of PKC by staurosporine partially reduced HG- but not BK-induced overexpression of these cytokines and that of VEGF. Staurosporine and PD98059 synergistically reduced the effect of HG on IL-6, CCL-2 and TGF-beta expression. The B(2)KR blocker, icatibant, downregulated BK- and HG-induced MAPK p42/p44 but not HG-induced PKC activation and partially reduced both HG- and BK-induced IL-6, CCL-2 and TGF-beta secretion. HG stimulated expression of KLK1 and low-molecular-weight kininogen (LMWK) and its downstream effects were attenuated by aprotinin (tissue kallikrein inhibitor). The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, rosiglitazone, attenuated HG-induced PKC but not HG- or BK- induced MAPK p42/44 activation and reduced HG-stimulated VEGF, along with IL-6, CCL-2 and TGF-beta secretion. Rosiglitazone plus icatibant further reduced these effects of HG. CONCLUSIONS: In conclusion, HG stimulates tubular proinflammatory, profibrotic and angiogenic signals, which is partly mediated through BK via MAPK signalling and partly through PKC independent of BK. The potential therapeutic role of complementary B(2)KR blockade and PPAR-gamma activation deserves clinical investigation.

Growth hormone treatment during hemodialysis in a randomized trial improves nutrition, quality of life, and cardiovascular risk.

Nutritional markers, such as lean body mass (LBM) and serum albumin, predict outcome in dialysis patients, in whom protein-energy malnutrition is associated with increased morbidity and mortality. The metabolic effects of human growth hormone (hGH) may improve the nutritional and cardiovascular health of these patients and consequently reduce morbidity and mortality. The aim of this study was to establish clinical proof of concept of hGH treatment for the improvement of the nutritional status in adult patients who are on maintenance hemodialysis. A total of 139 adult patients who were on maintenance hemodialysis and had serum albumin levels < or =40 g/L were randomly assigned to 6 mo of treatment with placebo or 20, 35, or 50 microg/kg per d hGH. Change in LBM and serum albumin (primary outcomes), health-related quality of life, and secondary efficacy and safety parameters were monitored. The study showed that hGH treatment increased LBM significantly at all dosage levels (2.5 kg [95% confidence interval 1.8 to 3.1] versus -0.4 kg [95% confidence interval -1.4 to 0.6]; P < 0.001 for pooled hGH groups versus placebo). Serum albumin tended to increase (P = 0.076), serum transferrin (P = 0.001) and serum HDL (P < 0.038) increased, and plasma homocysteine was reduced (P = 0.029). TNF-alpha also tended to decrease with treatment (P = 0.134). An improvement in the Role Physical SF-36 quality-of-life subscale was observed (P = 0.042). There were no differences in clinically relevant adverse events between groups. In conclusion, hGH therapy safely improves LBM, other markers of mortality and morbidity, and health-related quality of life in adult patients who are on maintenance hemodialysis. A long-term study is warranted to investigate whether these treatment benefits result in reduced mortality and morbidity.

Ganoderma extract prevents albumin-induced oxidative damage and chemokines synthesis in cultured human proximal tubular epithelial cells.

BACKGROUND: Ganoderma lucidum (Ganoderma or lingzhi) is widely used as an alternative medicine remedy to promote health and longevity. Recent studies have indicated that components extracted from Ganoderma have a wide range of pharmacological actions including suppressing inflammation and scavenging free radicals. We recently reported that tubular secretion of interleukin-8 (IL-8) induced by albumin is important in the pathogenesis of tubulointerstitial injury in the proteinuric state. In this study, we explored the protective effect of Ganoderma extract (LZ) on albumin-induced kidney epithelial injury. METHODS: Growth arrested human proximal tubular epithelial cells (PTECs) were incubated with 0.625 to 10 mg/ml human serum albumin (HSA) for up to 72 h. HSA induced DNA damage and apoptosis in PTEC in a dose- and time-dependent manner. Co-incubation of PTEC with 4-64 microg/ml LZ significantly reduced the oxidative damage and cytotoxic effect of HSA in a dose-dependent manner (P<0.001). Increased release of IL-8 and soluble intercellular adhesion molecules-1 (sICAM-1) in PTEC induced by HSA was ameliorated by co-incubation with Ganoderma (16 microg/ml). To explore the components of LZ that exhibited most protective effect in HSA-induced PTEC damages, LZ was further separated into two sub-fractions, LZF1 (MW <30 kDa) and LZF2 (MW <3 kDa), by molecular sieving using millipore membrane. PTEC were incubated with 5 mg/ml HSA in the presence of different doses of LZF1, LZF2 or unfractionated LZ. RESULTS: There was no difference in the degree of protection from HSA-induced cytotoxicity or oxidative DNA damage between different fractions of LZ. However, low molecular weight LZ (<3 kDa) was most effective in reducing sICAM-1 released from HSA-activated PTEC whereas the high molecular weight LZ (unfractionated LZ) was more effective in diminishing IL-8 production. CONCLUSIONS: Our results suggest that Ganoderma significantly reduces oxidative damages and apoptosis in PTEC induced by HSA. The differential reduction of IL-8 or sICAM-1 released from HSA-activated PTEC by different components of the LZ implicates that components of Ganoderma with different molecular weights could play different roles and operate different mechanisms in preventing HSA-induced PTEC damage.

The study of Chinese medicinal herbal formula Shen San Fang in the treatment of experimental IgA nephropathy.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis worldwide, yet there is no effective or specific therapy. Shen San Fang (S3F) is a traditional Chinese herbal medicinal formula that has been used in China for many years to treat patients with hematuria. The aim of this study is to test the therapeutic value of S3F in an experimental model of IgAN. IgAN was induced in Lewis rats by continuous oral immunization with bovine gamma-globulin (BGG) in the drinking water for 8 weeks, followed by intravenous injection of 1 mg BGG daily for 3 successive days. The rats were randomly divided into four groups (five rats/group): control, control receiving S3F, induction of IgAN, and IgAN receiving S3E S3F decoction was fed to rats beginning week 4 from the first day of oral sensitization with BGG. The S3F treatment was continued until the rats were sacrificed or for a 4-week period. Hematuria, renal immunohistochemistry for IgA and transforming growth factor-beta 1 (TGF-beta1), renal histopathology, and renal content of TGF-beta1 were measured. Rats developing IgAN had marked hematuria, profound mesangial proliferation and mesangial expansion, intense and diffuse glomerular IgA deposition, increased glomerular TGF-beta1 expression, and raised renal TGF-beta1 levels. S3F treatment resulted in a significant reduction of hematuria, decreased mesangial IgA deposition, weaker immunostaining of TGF-beta1 in glomerulus, and a lower renal TGF-beta1 concentration. Our animal data suggests a therapeutic value for the Chinese medicinal formula S3F in experimental IgAN. This beneficial effect was due to reduced glomerular IgA deposition and TGF-beta1 expression. Our preliminary findings hold promise for future human therapy.

In vitro study examining the effect of sub-lethal QS 755 nm lasers on the expression of p16INK4a on melanoma cell lines.

BACKGROUND AND OBJECTIVES: Q-switched lasers had been used in the treatment of lentigo maligna but their role remains controversial. While previous studies have addressed the change in adhesion molecule expression after sub-lethal laser damage, no study has addressed the impact of sub-lethal laser damage at a molecular level. The p16 gene has been proposed as the candidate gene for melanoma. Our objective is to examine the effect of sub-lethal laser damage on p16 expression in melanoma cell lines. STUDY DESIGN/MATERIALS AND METHODS: Three human melanoma cell lines-HTB 66, Sk-mel-24 (HTB 71), and G361-were irradiated by a Q-switched 755 nm Alexandrite laser at fluencies that ranged from 0.85 to 2.0 J/cm(2). HTB 66 was the only cell line with significant expression of p16INK4a while the other two cells lines were p16INK4a negative and served as negative control. Protein and mRNA expression for p16 were assessed by flow cytometry and RT-PCR, respectively. RESULTS: The level of p16INK4a protein in cell line HTB 66 increased significantly after laser irradiation as compared with non-irradiated cells. The level of p16INK4a protein did not change in p16INK4a-negative cell lines (Sk-mel-24 and G361). However, there was only a slight increase in the percentage of G0/G1 phase cells. CONCLUSIONS: Sub-lethal laser damage could increase DNA damage leading to an increase in p16 expression, and such effect would be particularly undesirable for patients with p16 mutation. Further studies are warranted to examine the role of sub-lethal laser damage in inducing p16 mutation.