Highlights from the Tenth International Workshop on HIV Persistence during Therapy, December 13-16, 2022, Miami, Florida-USA.

The International Workshop on HIV Persistence during Therapy provides a forum in which HIV/AIDS researchers gather to share the latest research findings related to viral reservoirs and cure. The Tenth Workshop, which was attended by over 400 delegates, extended over 4 days and comprised eight sessions covering topics from the basic science of viral persistence to therapeutic approaches to HIV cure. Furthermore, satellite sessions on the first day of the Conference featuring cure research endeavours being pursued by the Bill and Melinda Gates Foundation as well as those being coordinated under the National Institutes of Health Martin Delaney Collaboratory program, provided important updates on research advances being made in these initiatives. As with previous conferences, the International Workshop on HIV Persistence during Therapy is primarily abstract-driven with only one invited talk for each of the sessions. This format, therefore, increases the number of presentations from early-stage investigators. Furthermore, presentations by Community representatives illustrated approaches to creating cure research literacy with effective messaging for the Community. The following article offers a synopsis of the meeting sessions. Due to space constraints, some presentations may have only been briefly discussed. Nevertheless, the Workshop abstracts can be found online (https://www/sciencedirect.com/journal/journal-of-virus-eradication/vol/8/suppl/S).

IgG fractions from patients with antiphospholipid syndrome and systemic lupus erythematosus bind to platelets, but do not affect collagen-induced platelet activation.

Anti-beta-2 glycoprotein 1 (anti-ß2GP1) is an antiphospholipid antibody found in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Its presence commonly is associated with thrombosis; however, the mechanisms of interaction of anti-ß2GP1 antibodies and platelets remain unclear. We investigated the effects of APS and SLE patient-derived IgG fractions on collagen-mediated platelet aggregation and examined the binding of patient-derived IgG to platelets before and after activation by collagen. IgG fractions, 150, 200, 300 or 350 µg/ml, isolated from 11 patients with APS and SLE were incubated with two sets of platelet-rich plasma (PRP) in the incubation wells of an aggregometer. The first set was activated by collagen and the other set was incubated for an additional 10 min. All platelets were collected by centrifugation and fixed in cell blocks. We assessed binding of IgG to platelets using immunocytochemistry (ICC). Patient-derived IgG fractions did not affect collagen-induced platelet aggregation. ICC staining using anti-human IgG antibodies demonstrated that patient-derived IgG fractions had greater affinity for non-activated platelets than those activated by 0.75 µg/ml collagen. Patient-derived IgG fractions bound to the surface of platelets and potentially could be internalized by platelets. IgG fractions from APS and SLE patients may sensitize non-activated platelets, which could increase platelet reactivity and thrombotic risk in patients. We did not detect secondary effects of patient-derived IgG fractions.

Magnetic resonance cholangiogram patterns and clinical profiles of ketamine-related cholangiopathy in drug users.

BACKGROUND & AIMS: Recreational ketamine use has emerged as an important health and social issue worldwide. Although ketamine is associated with biliary tract damage, the clinical and radiological profiles of ketamine-related cholangiopathy have not been well described. METHODS: Chinese individuals who had used ketamine recreationally at least twice per month for six months in the previous two years via a territory-wide community network of charitable organizations tackling substance abuse were recruited. Magnetic resonance cholangiography (MRC) was performed, and the findings were interpreted independently by two radiologists, with the findings analysed in association with clinical characteristics. RESULTS: Among the 343 ketamine users referred, 257 (74.9%) were recruited. The mean age and ketamine exposure duration were 28.7 (±5.8) and 10.5 (±3.7) years, respectively. A total of 159 (61.9%) had biliary tract anomalies on MRC, categorized as diffuse extrahepatic dilatation (n = 73), fusiform extrahepatic dilatation (n = 64), and intrahepatic ductal changes (n = 22) with no extrahepatic involvement. Serum alkaline phosphatase (ALP) level (odds ratio [OR] 1.007; 95% CI 1.002-1.102), lack of concomitant recreational drug use (OR 1.99; 95% CI 1.11-3.58), and prior emergency attendance for urinary symptoms (OR 1.95; 95% CI 1.03-3.70) had high predictive values for biliary anomalies on MRC. Among sole ketamine users, ALP level had an AUC of 0.800 in predicting biliary anomalies, with an optimal level of ≥113 U/L having a positive predictive value of 85.4%. Cholangiographic anomalies were reversible after ketamine abstinence, whereas decompensated cirrhosis and death were possible after prolonged exposure. CONCLUSIONS: We have identified distinctive MRC patterns in a large cohort of ketamine users. ALP level and lack of concomitant drug use predicted biliary anomalies, which were reversible after abstinence. The study findings may aid public health efforts in combating the growing epidemic of ketamine abuse. LAY SUMMARY: Recreational inhalation of ketamine is currently an important substance abuse issue worldwide, and can result in anomalies of the biliary system as demonstrated by magnetic resonance imaging. Although prolonged exposure may lead to further clinical deterioration, such biliary system anomalies might be reversible after ketamine abstinence. Clinical trial number: NCT02165488.

Surface-enhanced Raman spectroscopy of novel psychoactive substances using polymer-stabilized Ag nanoparticle aggregates.

A set of seized "legal high" samples and pure novel psychoactive substances have been examined by surface-enhanced Raman spectroscopy using polymer-stabilized Ag nanoparticle (Poly-SERS) films. The films both quenched fluorescence in bulk samples and allowed identification of µg quantities of drugs collected with wet swabs from contaminated surfaces.

Extraction, characterization and application of malva nut gum in water treatment.

In view of green developments in water treatment, plant-based flocculants have become the focus due to their safety, degradation and renewable properties. In addition, cost and energy-saving processes are preferable. In this study, malva nut gum (MNG), a new plant-based flocculant, and its composite with Fe in water treatment using single mode mixing are demonstrated. The result presents a simplified extraction of the MNG process. MNG has a high molecular weight of 2.3 × 105 kDa and a high negative charge of -58.7 mV. From the results, it is a strong anionic flocculant. Moreover, it is observed to have a branch-like surface structure. Therefore, it conforms to the surface of particles well and exhibits good performance in water treatment. In water treatment, the Fe-MNG composite treats water at pH 3.01 and requires a low concentration of Fe and MNG of 0.08 and 0.06 mg/L, respectively, when added to the system. It is concluded that for a single-stage flocculation process, physico-chemical properties such as molecular weight, charge of polymer, surface morphology, pH, concentration of cation and concentration of biopolymeric flocculant affect the flocculating performance.

Does gestational diabetes mellitus affect respiratory outcome in late-preterm infants?

BACKGROUND: Both gestational diabetes mellitus (GDM) and late-preterm delivery at 34-36 weeks' gestation are independently associated with neonatal respiratory complications, but it is unknown whether their combination increases further its risk. We therefore appraised the independent effect of GDM on the respiratory outcome of late-preterm infants. METHODS: In a retrospective cohort study, respiratory outcome of 911 infants delivered at 34-36 weeks' gestation between 1 January 2009 and 30 August 2012 from mothers with GDM (study group, n=130) was compared with infants delivered at the same gestation by mothers without GDM (control group, n=781). RESULTS: The study group had significantly higher incidence of transient tachypnoea of newborn (TTN, p=0.02) and air leak (p=0.012), and required more respiratory support, including oxygen, continuous positive airway pressure (CPAP), mechanical ventilation and neonatal intensive care, with a longer length of hospital stay, but not duration on respiratory support. On logistic regression analysis, GDM is an independent risk factor for TTN (aOR=1.5, 95% C.I.1.0-2.4), CPAP (aOR=2.37, 95% C.I. 1.05-4.89), mechanical ventilation (aOR=4.02 95% C.I. 1.57-10.32) and neonatal intensive care (aOR 1.83, 95% C.I. 1.05-3.87). CONCLUSIONS: Our results demonstrated an independent effect of GDM on the risk of severe respiratory complications in late-preterm infants. Additional close monitoring and timely intervention are necessary in the management of these infants.

The regulation of cytotoxicity and cyclooxygenase-2 expression by 2-hydroxy-ethyl methacrylate in human osteoblasts are related to intracellular glutathione levels.

AIM: To investigate the effects of 2-hydroxy-ethyl methacrylate (HEMA) on cytotoxicity and cyclooxygenase-2 (COX-2) protein expression in human osteoblasts. METHODOLOGY: Cytotoxicity was judged using an Alamar Blue reduction assay on human osteoblast cell line U2OS. Western blot was used to evaluate the expression of COX-2 protein by HEMA. To determine whether glutathione (GSH) levels were important in cytotoxicity and COX-2 expression of HEMA, cells were pre-treated with the GSH precursor, 2-oxothiazolidine-4-carboxylic acid (OTZ), to boost thiol levels, or buthionine sulfoximine (BSO) to deplete GSH. Paired Student's t-tests were applied for the statistical analysis of the results. RESULTS: HEMA demonstrated a cytotoxic effect to U2OS cells in a dose-dependent manner (P < 0.05). The 50% inhibition concentration of HEMA was approximately 3 mmol L(-1) . HEMA was found to induce COX-2 protein expression in U2OS cells (P < 0.05). The addition of OTZ acted as a protective effect on HEMA-induced cytotoxicity and COX-2 expression (P < 0.05). In contrast, the addition of BSO enhanced HEMA-induced cytotoxicity and COX-2 expression (P < 0.05). CONCLUSION: Taken together, the levels of HEMA that were tested inhibited cell growth on U2OS cells. HEMA has a significant potential for periapical toxicity. The activation of COX-2 protein expression may be one of the mechanisms of HEMA-induced periapical inflammation. These inhibitory effects were associated with intracellular GSH levels.

Preparation of micron/submicron hybrid patterns via a two-stage UV-imprint technique and their dimensional effects on cell adhesion and alignment.

Cell adhesion, movement and proliferation on a biomaterial have been broadly explored and known to be induced by the morphology and structure of material surfaces. In order to explore the effects of hybrid structures (combination of micro- and nanofeatures on a pattern) on cell adhesion and alignment, a micro-featured mold was firstly prepared using partial UV-irradiation and the protruding top of the mold was then imprinted with nano-featured templates via successive UV irradiation. An oxygen inhibition effect was utilized in the course of UV curing and a two-step molding process, to form multiscale hybrid structures. The poly(dimethyl siloxane) (PDMS) replica of the hybrid mold was manufactured and employed to fabricate hybrid polymeric patterns for cell attachment. The underlying micro-feature was chosen to be a 25-µm-wide pattern and the nanostructures on the protrusions of the micropattern were different ruled nanogrooves, either parallel or perpendicular to the micro-featured pattern. In cell attachment measurement, 3T3 fibroblasts attached to poly(methyl methacrylate) (PMMA) samples seemed to be preferentially located on the recessed area of the hybrid patterns; however, 3T3 fibroblasts were aligned with nano-features, no matter if the nanogrooves were parallel or perpendicular to the micro-featured patterns. The nanogroove size was found to determine the effectiveness of cell alignment.

Heat shock protein 27 expression in areca quid chewing-associated oral squamous cell carcinomas.

OBJECTIVES: Heat shock protein (HSP) 27 is a low-molecular-weight protein that functions as a molecular chaperone and plays a cytoprotective role through its antioxidant activity during cell stress. Areca quid chewing is associated with the high incidence of oral squamous cell carcinomas (OSCCs) in Taiwan. The aim of this study was to compare heat shock protein 27 (HSP27) expression in OSCCs and the normal oral tissues. METHODS: Forty-eight OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry for HSP27. The normal human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot for HSP27. Furthermore, epigallocatechin-3 gallate (EGCG), glutathione precursor N-acetyl-L-cysteine (NAC), cyclooxygenase-2 inhibitor NS-398, HSP inhibitor quercetin, extracellular signal-regulated protein kinase (ERK) inhibitor PD98059, and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. RESULTS: Heat shock protein 27 exhibited higher expression in OSCCs than normal specimens (P < 0.05). Arecoline was found to elevate HSP27 expression in a dose- and time-dependent manner (P < 0.05). The additions of pharmacological agents were found to inhibit arecoline-induced HSP27 expression (P < 0.05). CONCLUSIONS: Heat shock protein 27 expression is significantly elevated in areca quid chewing-associated OSCCs. Arecoline-induced HSP27 expression was downregulated by EGCG, NS398, NAC, quercetin, PD98059, and SB203580.

Molecular analysis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency in Hong Kong Chinese patients.

BACKGROUND: Congenital adrenal hyperplasia (CAH) caused by 21-hydroxylase deficiency (21OHD) is an autosomal recessive disorder due to mutation in the CYP21A2 gene. OBJECTIVE: To elucidate the genetic basis of 21-hydroxylase-deficient CAH in Hong Kong Chinese patients. PATIENTS AND METHODS: Mutational analysis of the CYP21A2 gene was performed on 35 Hong Kong Chinese patients with 21OHD using direct DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULTS: The genetic findings of 21 male and 14 female patients are the following: c.293-13A/C>G (intron 2 splice site; 20 alleles), p.I172N (13), p.R356W (7), p.Q318X (4). A total of 20 mutant alleles contained gross deletion/conversion of all or part of the CYP21A2 gene. A novel mutation, c.1367delA (p.D456fs), was detected in one patient. One patient had only a heterozygous mutation detected. Out of 35 patients, 16 would have been incorrectly genotyped if either DNA sequencing or MLPA alone was used for molecular analysis. CONCLUSIONS: The frequency of various mutations in the studied patients differs from those reported in other Asian populations. Gross deletion/conversion accounts for nearly one-third of the genetic defects. Therefore, laboratories must include methods for detecting point mutations as well as gross deletions/conversions to avoid misinterpretation of genotype. Genotyping has increasingly been proven to be a useful tool for supplementing, if not replacing, hormonal profiling for the diagnosis of 21OHD.

Capsaicin in the periaqueductal gray induces analgesia via metabotropic glutamate receptor-mediated endocannabinoid retrograde disinhibition.

BACKGROUND AND PURPOSE: Capsaicin, an agonist of transient receptor potential vanilloid 1 (TRPV1) channels, is pro-nociceptive in the periphery but is anti-nociceptive when administered into the ventrolateral periaqueductal gray (vlPAG), a midbrain region for initiating descending pain inhibition. Here, we investigated how activation of TRPV1 channels in the vlPAG leads to anti-nociception. EXPERIMENTAL APPROACH: We examined synaptic transmission and neuronal activity using whole-cell recordings in vlPAG slices in vitro and hot-plate nociceptive responses in rats after drug microinjection into the vlPAG in vivo. KEY RESULTS: Capsaicin (1-10 µM) depressed evoked GABAergic inhibitory postsynaptic currents (eIPSCs) in vlPAG slices presynaptically, while increasing miniature excitatory PSC frequency. Capsaicin-induced eIPSC depression was antagonized by cannabinoid CB1 and metabotropic glutamate (mGlu5) receptor antagonists, and prevented by inhibiting diacylglycerol lipase (DAGL), which converts DAG into 2-arachidonoylglycerol (2-AG), an endocannabinoid. Capsaicin induced membrane depolarization in 2/3 neurons recorded but, overall, increased neuronal firings by increasing evoked postsynaptic potentials. Intra-vlPAG capsaicin reduced hot-plate responses in rats, effects blocked by CB1 and mGlu receptor antagonists. Effects of capsaicin were antagonized by SB 366791, a TRPV1 channel antagonist. CONCLUSIONS AND IMPLICATIONS: Capsaicin activated TRPV1s on glutamatergic terminals to release glutamate which activated postsynaptic mGlu5 receptors, yielding 2-AG from DAG by DAGL hydrolysis. 2-AG induces retrograde inhibition (disinhibition) of GABA release via presynaptic CB1 receptors. This disinhibition in the vlPAG leads to anti-nociception by activating the descending pain inhibitory pathway. This is a novel TRPV1 channel-mediated anti-nociceptive mechanism in the brain and a new interaction between vanilloid and endocannabinoid systems.

Hypoxia inducible factor-1α expression in areca quid chewing-associated oral squamous cell carcinomas.

OBJECTIVES: Hypoxia inducible factor (HIF)-1α gene expression is mainly induced by tissue hypoxia. Overexpression of HIF-1α has been demonstrated in a variety of cancers. The aim of this study was to compare HIF-1α expression in normal human oral epithelium and areca quid chewing-associated oral squamous cell carcinoma (OSCC) and further to explore the potential mechanisms that may lead to induce HIF-1α expression. METHODS: Twenty-five OSCC from areca quid chewing-associated OSCC and 10 normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry. The oral epithelial cell line GNM cells were challenged with arecoline, a major areca nut alkaloid, by using Western blot analysis. Furthermore, glutathione precursor N-acetyl-l-cysteine (NAC), AP-1 inhibitor curcumin, extracellular signal-regulated protein kinase inhibitor PD98059, and protein kinase C inhibitor staurosporine were added to find the possible regulatory mechanisms. RESULTS: Hypoxia inducible factor-1α expression was significantly higher in OSCC specimens than normal specimen (P<0.05). Arecoline was found to elevate HIF-1α expression in a dose- and time-dependent manner (P<0.05). The addition of NAC, curcumin, PD98059, and staurosporine markedly inhibited the arecoline-induced HIF-1α expression (P<0.05). CONCLUSIONS: Hypoxia inducible factor-1α expression is significantly upregulated in areca quid chewing-associated OSCC and HIF-1α expression induced by arecoline is downregulated by NAC, curcumin, PD98059, and staurosporine.

Adult neurogenesis is reduced in the dorsal hippocampus of rats displaying learned helplessness behavior.

Clinical and preclinical studies suggest that the hippocampus has a role in the pathophysiology of major depression. In the learned helplessness (LH) animal model of depression after inescapable shocks (ISs) animals that display LH behavior have reduced cell proliferation in the hippocampus; this effect can be reversed by antidepressant treatment. Using this model, we compared rats that displayed LH behavior and rats that did not show LH behavior (NoLH) after ISs to determine whether reduced hippocampal cell proliferation is associated with the manifestation of LH behavior or is a general response to stress. Specifically, we examined cell proliferation, neurogenesis, and synaptic function in dorsal and ventral hippocampus of LH and NoLH animals and control rats that were not shocked. The LH rats had showed reduced cell proliferation, neurogenesis, and synaptic transmission in the dorsal hippocampus, whereas no changes were seen in the ventral hippocampus. These changes were not observed in the NoLH animals. In a group of NoLH rats that received the same amount of electrical shock as the LH rats to control for the unequal shocks received in these two groups, we observed changes in Ki-67(+) cells associated with acute stress. We conclude that reduced hippocampal cell proliferation and neurogenesis are associated with the manifestation of LH behavior and that the dorsal hippocampus is the most affected area.

Regulation of type I plasminogen activator inhibitor in human gingival fibroblasts with cyclosporine A.

OBJECTIVES: Cyclosporine A (CsA) is used as an immunosuppressive agent and its prominent side effect is the induction of gingival overgrowth. Type I plasminogen activator inhibitor (PAI-1) has shown to play an important role in CsA-induced gingival overgrowth. However, little is known about whether factors can modulate CsA-induced PAI-1 expression. METHODS: Cytotoxicity, reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay were used to investigate the effects of Human gingival fibroblasts (HGFs) exposed to CsA. In addition, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, interlukin-1alpha, tumor necrosis factor-alpha, mitogen-activated protein kinase kinase (MEK) inhibitor U0126, signal-regulated protein kinase (ERK) inhibitor PD98059 and cell-permeable glutathione precursor N-acetyl-L-cysteine (NAC) were added to test how they modulated the effects of CsA-induced PAI-1 expression. RESULTS: The concentration of CsA higher than 500 ng ml(-1) demonstrated cytotoxicity to HGFs (P < 0.05). Periodontal pathogens as well as proinflammatory cytokines were found to increase the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). Pharmacological agents NAC, U0126, and PD98059 were found to decrease the CsA-induced PAI-1 mRNA and protein expression (P < 0.05). CONCLUSIONS: Cyclosporine A (CsA) may predispose to gingival overgrowth under inflammatory environments. The regulation of PAI-1 expression induced by CsA might be critically related with the intracellular glutathione and the ERK-MAPK pathway.

Disseminated Cryptococcus neoformans var. grubii infections in intensive care units.

A retrospective study of clinical characteristics, outcome and prognostic factors of patients with cryptococcosis was undertaken in intensive care units (ICUs) of a medical centre for the period 2000-2005. Twenty-six patients with Cryptococcus neoformans var. grubii infection were identified (16 males, median age 58 years). The most frequent underlying diseases were liver cirrhosis (38.5%), diabetes mellitus (26.9%) and HIV infection (19.2%). The most frequently identified sites of infection were blood (61.5%), cerebrospinal fluid (38.5%) and airways (34.6%). The mean Acute Physiologic and Chronic Health Evaluation II score at ICU admission was 22.46. The ICU mortality rate in these patients was 73.1% (19/26) and there were a further two mortalities recorded after discharge from ICU, reaching a total mortality rate of 80.8% (21/26). Patients with ICU survival >2 weeks had lower rates of HIV infection (P=0.004), less use of inotropic agents during ICU stay (P<0.001) and lower white blood cell counts (P=0.01). After adjusting for clinical variables in the multivariate Cox regression model, diabetes and cryptococcal infection after ICU admission were independent predictors of good long-term prognosis (P=0.015) and HIV infectious status was associated with poor outcome (P=0.012).

Characterization of biopolymeric flocculant (pectin) and organic synthetic flocculant (PAM): a comparative study on treatment and optimization in kaolin suspension.

Polyacrylamide (PAM), a commonly used organic synthetic flocculant, is known to have high reduction in turbidity treatment. However, PAM is not readily degradable. In this paper, pectin as a biopolymeric flocculant is used. The objectives are (i) to determine the characteristics of both flocculants (ii) to optimize the treatment processes of both flocculants in synthetic turbid waste water. The results obtained indicated that pectin has a lower average molecular weight at 1.63 x 10(5) and PAM at 6.00 x 10(7). However, the thermal degradation results showed that the onset temperature for pectin is at 165.58 degrees C, while the highest onset temperature obtained for PAM is at 235.39 degrees C. The optimum treatment conditions for the biopolymeric flocculant for flocculating activity was at pH 3, cation concentration at 0.55 mM, and pectin concentration at 3 mg/L. In contrast, PAM was at pH 4, cation concentration >0.05 mM and PAM concentration between 13 and 30 mg/L.

Analysis and optimization of flocculation activity and turbidity reduction in kaolin suspension using pectin as a biopolymer flocculant.

The performance of pectin in turbidity reduction and the optimum condition were determined using Response Surface Methodology (RSM). The effect of pH, cation's concentration, and pectin's dosage on flocculating activity and turbidity reduction was investigated at three levels and optimized by using Box-Behnken Design (BBD). Coagulation and flocculation process were assessed with a standard jar test procedure with rapid and slow mixing of a kaolin suspension (aluminium silicate), at 150 rpm and 30 rpm, respectively, in which a cation e.g. Al(3+), acts as coagulant, and pectin acts as the flocculant. In this research, all factors exhibited significant effect on flocculating activity and turbidity reduction. The experimental data and model predictions well agreed. From the 3D response surface graph, maximum flocculating activity and turbidity reduction are in the region of pH greater than 3, cation concentration greater than 0.5 mM, and pectin dosage greater than 20 mg/L, using synthetic turbid wastewater within the range. The flocculating activity for pectin and turbidity reduction in wastewater is at 99%.

Up-regulation of osteolytic mediators in human osteosarcoma cells stimulated with nicotine.

BACKGROUND AND OBJECTIVE: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. However, little is known about how nicotine influences the expression of osteolytic mediators in cigarette smoking-associated periodontal diseases. The aim of this study was to investigate the expression of interleukin-1, interleukin-8, receptor activator of nuclear factor-kappaB ligand (RANKL), gelatinases and tissue-type plasminogen activator in U2OS cells (from the human osteosarcoma cell line) stimulated with nicotine. MATERIAL AND METHODS: Differences in the expression of interleukin-1, interleukin-8 and RANKL mRNAs, in response to exposure to various concentrations of nicotine (0, 0.125, 0.25, 0.5 and 1 mm) were evaluated in U2OS cells using the reverse transcription-polymerase chain reaction.In addition, the levels of interleukin-1, interleukin-8 and RANKL proteins were determined using enzyme-linked immunosorbent assays. The gelatinolytic and caseinolytic activities in nicotine treated-U2OS cells were demonstrated using gelatin and casein zymography, respectively. RESULTS: Nicotine was found to increase the expression of interleukin-1, interleukin-8 and RANKL mRNA and protein in U2OS cells (p < 0.05). The gelatin zymograms revealed that matrix metalloproteinase (MMP)-2 and MMP-9 were secreted by U2OS cells. The secretion of MMP-2 and MMP-9 occurred in a dose-dependent manner that was dependent on the concentration of nicotine (p < 0.05). Casein zymography exhibited a caseinolytic band with a molecular weight of 70 kDa, indicative of the presence of tissue-type plasminogen activator. Tissue-type plasminogen activator was also found to be up-regulated by nicotine in a dose-dependent manner (p < 0.05). CONCLUSION: Taken together, the results of the present study indicated that smoking modulation of bone destruction in periodontal disease may involve various osteolytic mediators, such as interleukin-1, interleukin-8, RANKL, MMP-2, MMP-9, and tissue-type plasminogen activator.

Charcot foot in a Hong Kong Chinese diabetic population.

OBJECTIVES: To delineate the epidemiology of Charcot foot in Hong Kong Chinese diabetic patients, and to provide baseline data for benchmarking the clinic service for this special group of patients. DESIGN: Retrospective cohort study. SETTING: Regional hospital, Hong Kong. PATIENTS: Diabetic patients with Charcot foot and age- and sex-matched diabetic foot clinic attendees between 1995 and 2007. MAIN OUTCOME MEASURES: Clinical presentations were compared in patients with Charcot foot and the controls. RESULTS: Twenty-five patients were diagnosed with Charcot foot over 12 years; 60% were male. At the time of diagnosis, the mean age was 59 (standard deviation, 14; range, 38-85) years, with diabetes being diagnosed for a mean of 11 (standard deviation, 8; range, 0-30) years. Retinopathy was noted in 36% (n=9) and nephropathy in 20% (n=5) of the Charcot foot patients. No patient had peripheral vascular disease. This finding was statistically significant. Delayed presentation occurred in 11 patients. Presentation was usually unilateral. In the minority (n=3, 12%) with bilateral involvement, presentation was sequential. Charcot arthropathy affected the mid-foot in 64% of the patients. Superimposed infection was common (61%). Recurrent ulceration occurred in 11%, all of whom presented late. Only one patient underwent major amputation, but the 5-year mortality of Charcot foot patients could be up to 33%. CONCLUSION: Charcot foot was uncommon in this population. Late presentation was common and might be related to superimposed infection; such patients were prone to recurrent ulcers.