Metabolic syndrome, endothelial injury, and subclinical atherosclerosis in patients with systemic lupus erythematosus.

OBJECTIVES: To study the link between metabolic syndrome (MetS), endothelial injury, and atherosclerosis in patients with systemic lupus erythematosus (SLE). METHODS: Consecutive SLE patients without a history of arterial thrombosis were screened for atherosclerosis at the carotid and coronary arteries by B-mode ultrasound [intima-media thickness (IMT)] and multidetector computed tomography (MDCT) scan (Agatston calcium scores), respectively. Plasma levels of homocysteine, high-sensitivity C-reactive protein (hsCRP), soluble vascular cell adhesion molecule (sVCAM)-1, P-selectin, and soluble thrombomodulin (sTM) were assayed. Patients were stratified according to the National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATP III) criteria for MetS, using the Asian criteria for abdominal obesity. Risk factors for atherosclerosis were studied. RESULTS: Of the 123 SLE patients (93% women; age 47.9+/-11 years; SLE duration 10.9+/-7.0 years) studied, 20 (16.3%) had MetS. The prevalence of MetS in the SLE patients was significantly higher than in 492 age- and sex-matched healthy controls (9.6%; p=0.03). Coronary calcification and abnormal carotid IMT were detected in 38 (31%) and 72 (59%) of SLE patients, respectively. Patients with MetS had a significantly higher Agatston score (69.5+/-95 vs. 16.4+/-57; p=0.03) and a numerically higher carotid IMT (p=0.43) than those without. In a logistic regression model, the MetS [odds ratio (OR) 3.11, 95% confidence interval (CI) 1.01-9.59, p=0.049] was associated with coronary atherosclerosis after adjustment for age and other risk factors. In addition, patients with MetS had significantly higher levels of hsCRP (p=0.002), homocysteine (p=0.03), and sTM (p=0.01). CONCLUSIONS: The MetS is more prevalent in SLE patients than the general population and is associated with endothelial injury and coronary atherosclerosis. More aggressive control of risk factors is justified in these patients.

Phosphatase and tensin homologue deleted on chromosome ten (PTEN) as a molecular target in lung epithelial wound repair.

BACKGROUND AND PURPOSE: Epithelial injury contributes to lung pathogenesis. Our work and that of others have identified the phosphoinositide-3 kinase (PI3K)/Akt pathway as a vital component of survival in lung epithelia. Therefore, we hypothesized that pharmacological inhibition of PTEN, a major suppressor of this pathway, would enhance wound closure and restore lung epithelial monolayer integrity following injury. EXPERIMENTAL APPROACH: We evaluated the ability of two bisperoxovanadium derivatives, bpV(phen) and bpV(pic), in differentiated primary human airway epithelia and BEAS2B cultures for their ability to inhibit PTEN, activate the PI3K/Akt pathway and restore epithelial monolayer integrity following mechanical injury. KEY RESULTS: BpV(phen) and bpV(pic) induced Akt phosphorylation in primary and BEAS2B cells in a dose and time dependent manner. Minimal toxicity was observed as measured by lactate dehydrogenase (LDH) release. To verify that Akt phosphorylation is specifically induced by PTEN inhibition, the PTEN positive cell line, DU145, and two PTEN negative cell lines, LNCaP and PC3, were examined. PTEN positive cells demonstrated a dose responsive increase in Akt phosphorylation whereas PTEN negative cells showed no response indicating that bpV(phen) directly suppresses PTEN without affecting auxiliary pathways. Next, we observed that exposure to either compound resulted in accelerated wound closure following mechanical injury. Similar effects were observed after transfection with a dominant negative isoform of PTEN and PTEN specific siRNA. CONCLUSIONS AND IMPLICATIONS: From these studies, we conclude that PTEN is a valid target for future studies directed at restoring epithelial barrier function after lung injury.

Quantification of substance P mRNA in human mononuclear phagocytes and lymphocytes using a mimic-based RT-PCR.

We have recently demonstrated that human monocytes and lymphocytes express the substance P (SP) gene at both the mRNA and protein level [Ho, W.Z., Lai, J.P., Zhu, X.H., Uvaydova, M., Douglas S.D., 1997. Human monocytes and macrophages express substance P and neurokinin-1 receptor. Journal of Immunology, 159, p. 5654; Lai, J.P., Douglas, S. D., Ho, W.Z., 1998. Human lymphocytes express substance P and its receptor. Journal of Neuroimmunology, 86, p. 80; Lai, J.-P., Douglas, S.D., Rappaport, E., Wu, J., Ho, W.-Z., 1998. Identification of a delta isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes. Journal of Neuroimmunology, 91, p. 121]. Using RT-PCR assay with several specific human SP primer pairs, we were able to differentiate four isoforms of preprotachykinin (PPT-A, the SP precursor) mRNA transcripts on ethidium bromide-stained agarose gels and clone the PCR amplified cDNA of the four isoforms (alpha, beta, gamma, and delta) of the PPT-A gene. In an effort to quantitatively measure PPT-A mRNA levels, we have developed a mimic-based RT-PCR assay to analyze total PPT-A mRNA levels in human monocytes and lymphocytes. We designed a specific human SP primer pair (HSP4/HSP3) to amplify a single fragment of cDNA derived from all four isoforms of PPT-A mRNA transcripts, with a sensitivity of 120 molecules per reaction. Thus the PPT-A mRNA transcripts in an unknown sample can be quantitatively analyzed using the mimic-based RT-PCR. The accuracy and reproducibility of this assay were confirmed by the plasmids containing alpha, beta, gamma and delta cDNA inserts and by in vitro synthesized mRNA from a plasmid containing beta isoform cDNA insert. Our data indicate that the SP mimic-based RT-PCR assay has potential advantages in studies of SP levels in a variety of human cells as well as in clinical specimens.

Human lymphocytes express substance P and its receptor.

We present data demonstrating the gene expression of substance P (SP) and its receptor in human peripheral blood-isolated lymphocytes. Using reverse transcribed polymerase chain reaction (RT-PCR) assay, preprotachykinin-A (substance-P) mRNA is detected in human peripheral blood-isolated lymphocytes. Among the alpha, beta, and gamma transcripts of the SP gene, only the beta and gamma transcripts are detectable in these cells. These RT-PCR amplified transcripts are recognized by Southern blot assay using a specific SP probe. Direct DNA sequence analysis of the RT-PCR products from lymphocytes also confirmed the structure of these transcripts which are identical to those found in human neuronal cells. At the protein level, human lymphocytes produced endogenous SP as determined by an enzyme immunoassay. Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released preformed SP from lymphocytes. In addition, using RT/nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (the receptor for SP) in human peripheral blood-isolated lymphocytes, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human lymphocytes express SP and its receptor support the notion that SP is biologically involved in regulating the functions of these cells in an autocrine fashion.

Identification of a delta isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes.

We have characterized preprotachykinin (PPT-A) gene transcript splicing products and identified a fourth isoform of PPT-A mRNA transcript in human peripheral blood-isolated monocytes and PBL. Using RT-PCR, Southern blot analysis and nucleotide sequencing analysis, we have identified the four isoforms of PPT-A transcripts (alpha, beta, gamma and delta) in human peripheral blood-isolated monocytes and PBL. The delta-PPT transcript present in the immune cells lacks exons 4 and 6. The sequences of exons 3, 5 and 7 of delta-PPT transcript completely match those of beta-PPT transcript. The alpha-PPT and beta-PPT sequences in these cells are identical to those obtained by Tan and Too (GenBank accession number U37539) and Harmar et al. (Genbank accession number X54469), but differ by a single nucleotide from another entry by Chiwakata et al. (Genbank accession number M68906). In comparison to this latter sequence, there was a C-->T change at amino acid position 87 (CCT-->CTT) which may result in a Pro to Leu change. Identification of the new SP mRNA transcript in both human CNS and immune cells supports the concept of an important biological link between CNS and immune system.

Substance P C-terminal octapeptide analogues augment tumor necrosis factor-alpha release by human blood monocytes and macrophages.

We have investigated the effects of the substance P C-terminal octapeptide analogues [Pro4, Glu (OBzl)11] SP4-11, [Hyp4, Glu(OBzl)11] SP4-11, [cHyp4, Glu(OBzl)11] SP4-11 and [kPro4, Glu(OBzl)11] SP4-11 on the constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF-alpha) in both freshly isolated human blood monocytes (FIBM) and monocyte-derived macrophages (MDM). The cells were treated with substance P and the substance P analogues at various concentrations (10-14 to 10-6 M) in the presence or absence of LPS and culture supernatants were analyzed for TNF-alpha as measured by an enzyme immunosorbent assay (ELISA). Monocytes and macrophages treated with the substance P analogues alone increased TNF-alpha secretion at a magnitude similar to the effect of entire undecapeptide substance P. The stimulatory effects of the substance P analogues on TNF-alpha secretion are inhibited by substance P antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-9-Leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the substance P receptor), indicating that these effects are specific and substance P receptor-mediated. Treatment of monocytes and macrophages with the substance P analogues in combination with LPS, however, showed no synergistic interaction in upregulation of TNF-alpha. These data indicate that the biological effect of substance P on TNF-alpha production by human monocytes and macrophages depends mainly on the sequence of the C-terminal region of the molecule.

Detection of substance P and its receptor in human fetal microglia.

Substance P, the most abundant neurokinin in the CNS, is a major modulator of the immune system. We have examined the gene expression of substance P and its receptor in human fetal brain microglia. Using reverse transcription-polymerase chain reaction and Southern blotting assay, the four isoforms of preprotachykinin-A gene transcripts (alpha, beta, gamma and delta) were detected in the microglia. The human fetal microglia produced significantly higher levels of endogenous substance P protein (640-850 pg/10(6) cells) than did human peripheral blood monocyte-derived macrophages (25-50 pg/10(6) cells), as determined by an enzyme immunoassay. Using immunohistochemical staining with an anti-substance P antibody, cell membrane substance P immunoreactivity was observed. In addition, we identified the presence of messenger RNA for neurokinin-1 receptor, a primary receptor for substance P in human fetal microglia.From these data, we propose that substance P and its receptor are biologically involved in regulating the functions of microglia, and potentially play an important role in host defense of the central nervous system.

Inhibition of HIV type 1 replication in chronically infected monocytes and lymphocytes by retrovirus-mediated gene transfer of anti-Rev single-chain variable fragments.

We investigated a strategy for gene therapy, intracellular expression of anti-HIV-1 Rev single-chain variable fragments (SFvs), in promonocytic (U1) and T (ACH-2) cell lines latently infected with HIV-1. The cellular and molecular mechanisms leading to activation of latent integrated HIV-1 provirus in U1 and ACH-2 cells have been well delineated. These cells produce HIV-1 in response to stimulation with certain cytokines. U1 and ACH-2 cells were transduced with a murine retroviral shuttle vector that expresses anti-Rev SFv (pLXSN-D8SFv-Rev) or with a control murine leukemia virus (MLV) vector (pLXSN). Tumor necrosis factor alpha (TFNalpha)-, interleukin 6 (IL-6)-, and phorbol myristate acid (PMA)-induced HIV-1 expression, as determined by reverse transcriptase (RT) assay, was significantly inhibited in cells transduced with pLXSN-D8SFv-Rev, compared with cells transduced with pLXSN. In addition, pLXSN-D8SFv-Rev-transduced cells, when incubated with monokine-enriched supernatants of human peripheral blood monocyte cultures, produced significantly less HIV-1 than did cells transduced with pLXSN. This resistance to cytokine-induced HIV-1 expression was demonstrated in SFv-transduced U1 and ACH-2 cells maintained in G418-free medium for 2 months. These data suggest that feasibility of utilizing various anti-HIV-1 SFvs to block activation of HIV-1 infection in vivo.

Microinvasive Nd:YAG laser therapy of early glottic carcinoma and its effect on soluble interleukin-2 receptor, interleukin-2, and natural killer cells.

OBJECTIVE: To investigate the effectiveness of microinvasive Nd:YAG laser therapy in human glottic Tis and T1 carcinomas, as well as its effect on the cellular immune function of the tumor-bearing hosts. STUDY DESIGN: We treated 34 patients with microinvasive Nd:YAG laser therapy and evaluated its effect on the cellular immune function of the host. METHODS: Thirty-four patients with glottic Tis or T1 squamous cell carcinoma were treated with fiberoptic laryngoscopic Nd:YAG laser surgery. Both before and after therapy, serum levels of soluble interleukin-2 receptor (SIL-2R) and interleukin-2 (IL-2), as well as natural killer (NK) cell activity, were determined by means of double-antibody sandwich technique, tritiated thymidine-deoxyribonucleoside incorporation, and iodine 125-uridine-deoxyribonucleoside release technique, respectively. RESULTS: All 34 patients tolerated the procedure well. A 3- to 7-year follow-up in a subgroup of 27 patients resulted in an estimated cure rate of 85.2% (23 of 27 patients). In all 27 patients with a regular follow-up, a subjective improvement of phonation was noted after therapy to various degrees. In 74% (20 of 27 patients), voice and speech subjectively recovered to almost normal levels. The post-therapy serum levels of SIL-2R were significantly declined (P <.001), whereas those of IL-2 and the NK activity were significantly elevated (P <.001) as compared with those detected before therapy. CONCLUSIONS: Therapy with fiberoptic laryngoscopic Nd:YAG laser surgery is simple, safe, effective and only minimally invasive for patients with glottic Tis or T1 carcinoma. At the same time, it has an immunoenhancing effect on its host.

The effect of shoulder-girdle loading by a school bag on lung volumes in Chinese primary school children.

BACKGROUND: Heavy loading of the spine may induce musculoskeletal problems in children. Local surveys reported frequent overloading of school bags carried by primary school children. The effect of an overweight school bag on the child's lung function has not been reported. AIMS: To investigate the effect of shoulder-girdle loading on forced expiratory lung volumes in primary school children and to compare this effect with that of an assumed kyphotic posture. STUDY DESIGN AND SUBJECTS: Forty-three primary school children, mean age 9.6 years underwent spirometry lung-function measurements, while adopting the following five conditions in random order: free standing; kyphotic standing; standing wearing a backpack weighing 10%, 20% and 30% of their body weight. OUTCOMES MEASURES: Forced expiratory volume in the first second (FEV1), forced vital capacity (FVC) and peak expiratory flow rate (PEF). RESULTS: There were no significant differences in FEV1 and FVC between free standing and the 10% body weight load. However, both FEV1 and FVC decreased significantly when the student adopted the kyphotic posture and when the load in the backpack was increased to 20% and 30% of body weight. CONCLUSIONS: This study demonstrates a restrictive effect on lung volumes when a school-bag load is heavier than 10% of a child's body weight. Our results also confirm the detrimental effect of a kyphotic posture on pulmonary mechanics and the necessity for health-care professionals to advocate proper postural advice to school children, teachers and parents.

Endoscopically assisted reconstruction of orbital medial wall fractures.

Traditional surgical approaches to orbital medial wall fractures are either direct extraocular skin incisions or indirect bicoronal flap. However, these methods can leave remarkable orbital scars or scalp alopecia with the possible associated complications. A minimally invasive endoscopic technique with the assistance of a 2.7-mm, 30-degree telescope through a medial transconjunctival incision has been successfully used to reconstruct the orbital medial wall fractures. This technique was applied to four patients who had orbital medial wall fractures. Three patients also had concomitant orbital floor fractures. The other had associated superior orbital fissure syndrome. All patients were presented with limited eye movement, positive forced duction test, horizontal diplopia, and enophthalmos (3 mm to 6 mm) preoperatively. The entrapped periorbital tissues in the ethmoid sinus were completely reduced endoscopically. The bone defect of orbital medial wall was reconstructed with autogenous rib bone grafts under endoscopic control. The patients were followed up for 8 to 16 months with an average of 11 months. Three patients recovered completely without any residual eye symptoms after intervention. Clinically significant residual enophthalmos of 3 mm occurred in the patient with the superior orbital fissure syndrome. His eye movement limitation caused by entrapment of medial rectus muscle was relieved postoperatively. There was no donor-site morbidity or any complications related to the endoscopically assisted procedure. Endoscopically assisted medial transconjunctival approach to the orbital medial wall fractures is an excellent adjunct for the exposure and complete reduction of herniated periorbital tissue and bony reconstruction of the medial orbital wall.

Endoscopically assisted mandibular subcondylar fracture repair.

The endoscope has been widely used in aesthetic surgery in recent years, but rarely has it been used in cases of facial trauma. From July of 1996 to December of 1996, the endoscope was used successfully to assist in the repair of mandibular subcondylar fractures in eight patients (five men and three women). Their ages ranged from 15 to 60 years with an average age of 31 years. Six of the patients had other associated mandibular fractures including angular, parasymphyseal, and contralateral subcondylar fractures. A 4.0-mm, 30-degree telescope was introduced to visualize the fracture site by means of an intraoral incision over the ascending ramus. A miniplate was used to stabilize the fracture site with the help of a percutaneous trocar. Intermaxillary fixation was applied for 3 to 6 days. Functionally, all patients returned to normal range of motion within 8 weeks. A slight deviation to the trauma site was noted on maximal opening in three patients, but this condition returned to normal 3 months after surgery. There was no facial palsy or lip numbness. The benefits of the endoscopic approach include not only the provision of better visualization and precise anatomic alignment of bony segments but also the avoidance of large facial scars and facial nerve injuries.

Effect of photodynamic therapy on selected laboratory values of patients with nasopharyngeal carcinoma.

We determined pre- and post-photodynamic therapy (PDT) serum levels of soluble interleukin-2 receptor (SIL-2R) and interleukin-2 (IL-2), as well as activity of natural killer (NK) cells, among 24 patients with either persistent or recurrent nasopharyngeal carcinoma (NPC), using a double-antibody sandwich technique, tritiated thymidine-deoxyribonucleoside incorporation, and iodine 125-uridine-deoxyribonucleoside release techniques, respectively. The results showed that the post-PDT serum level of SIL-2R had significantly declined (p < .0005), while that of IL-2 and the NK cell activity had significantly increased (p < .0005), compared with pre-PDT values, suggesting an immunoenhancing effect of PDT on NPC patients.

Management of carbon monoxide poisoning using oxygen therapy.

The management of carbon monoxide poisoning requires an accurate assessment of the extent of blood oxygenation. Measuring the fractional oxyhaemoglobin content by using co-oximetry gives a true picture of the oxygen-carrying capacity of blood in the presence of carboxyhaemoglobin. The use of readings from pulse oximetry or a standard blood gas analyser is insufficient and can be misleading. We report on a case of carbon monoxide poisoning to illustrate this potential pitfall.

Macroprolactin-a cause of pseudohyperprolactinaemia.

Macroprolactin is a complex of immunoglobulin G and monomeric prolactin with little biological activity in vivo. Macroprolactin cross-reacts in modern commercial prolactin assays, however, leading to pseudohyperprolactinaemia. This report is of three patients with macroprolactinaemia and the untoward consequences if this benign condition is misdiagnosed as genuine hyperprolactinaemia are discussed. One adult and one child without symptoms of hyperprolactinaemia were incidentally found to have elevated serum prolactin levels, one of whom had a pituitary incidentaloma. Repeat prolactin measurement after polyethylene glycol precipitation showed that the majority of circulating prolactin was macroprolactin. The third patient had galactorrhoea and pituitary microadenoma. Polyethylene glycol study showed that macroprolactinaemia exists simultaneously with genuine hyperprolactinaemia leading to falsely high serum prolactin levels. The recognition of this relatively common and benign condition is important in order to avoid misdiagnosis and unnecessary investigations and treatment. Particular attention must be paid to patients in whom the clinical and radiological findings are incompatible.

A survey of congenital heart disease in patients with oral clefts.

The purpose of this study is to determine the prevalence of congenital heart disease in patients with cleft lip and/or palate. We undertook a retrospective study of 1148 cases, age < 15 years old, with cleft lip and/or palate from January 1991 to December 1998, of which congenital heart disease was associated in 62 patients. The overall prevalence of congenital heart disease in patients with clefts was 5.4%. Of the 62 patients, there were 38 boys and 24 girls. We classified clefts into one of three categories; group 1: cleft lip alone; group 2: cleft lip and palate; group 3: cleft palate alone. The cleft lip and palate (group 2) was present in the majority (27 of 62; 44%) of patients with congenital heart disease. Twenty-five patients (40%) had cleft palate, and ten patients (16%) had cleft lip. Isolated atrial septal defect and ventricular septal defect are the two common congenital defects, which presented 23% and 21% of patients, respectively. Apart from congenital heart disease and cleft lip or palate, 56% (35 of 62 patients) and additional abnormalities. Central nervous system and skeletal malformations were the most common associated abnormalities. In our study, congenital heart disease was more common in patients of group 2 and group 3 than of group 1. In addition, there was a significantly greater proportion of patients associated with other systemic anomalies in groups 2 and 3 than in group 1 (chi-square chi 2 = 7.535, p = 0.023), but no significant difference was noted between group 2 and group 3. We recommend that it would be appropriate for all cleft patients to receive a routine examination for associated anomalies by a pediatrician. With the widespread use of echocardiography and/or brain sonography, the early diagnosis and treatment of these anomalies are possible.

Functional outcomes following surgical treatment of bilateral mandibular condylar fractures.

Debate continues regarding unilateral or bilateral treatment for mandibular condylar fractures. This retrospective study evaluates the functional outcomes of bilateral condylar process fractures after surgical intervention. From May 1994 to December 2004, 51 adult patients with bilateral mandibular condylar process fractures were studied. There were 33 cases of bilateral condylar fractures (type I); 12 cases of condylar-subcondylar fractures (type II); and six cases of bilateral subcondylar fractures (type III). All patients underwent open reduction and internal fixation. Four patients had chin deviation, six had malocclusion, three had poor chewing function and eight had limited mouth opening. Type I patients had a significantly higher incidence of limited mouth opening (P=0.039) and associated maxillary fractures (n=12) and psychiatric disease (n=6) which yielded significantly poor functional outcomes. Complications included transient facial paresis (n=4), fracture and loosening of postoperative plates (n=3) and surgical wound infections (n=2). Open reduction with rigid fixation for bilateral condylar fractures provided satisfactory functional outcomes in this study. Concomitant maxillary fractures and underlying psychiatric problems are poor outcome factors. Aggressive rehabilitation in the first 9 months is important for early functional recovery.

Analytical and biological considerations in the measurement of cell-associated CCR5 and CXCR4 mRNA and protein.

The accurate measurement of T cell-associated CC chemokine receptor type 5 (CCR5) and CXC chemokine receptor type 4 (CXCR4) expression, including expression of CCR5 and CXCR4 mRNA as an immune measure of immunologic response to highly active antiretroviral therapy (HAART) and newer agents, including entry inhibitors, is essential. Previous studies have reported alterations in lymphocyte cell membrane CCR5 expression that were related to blood collection and cell separation media. Clinical trials often require the transport of specimens to central laboratories for evaluation, resulting in significant time delays between specimen procurement and analysis. This study shows that CCR5 expression on naïve and memory T cells is influenced by blood collection media and specimen age. Peripheral blood collected in Streck Vacutainer tubes containing a cell stabilizer and fixative was found to improve detection of CCR5 expression compared to specimens collected in K2 EDTA anticoagulant. The selection of flow cytometry gating strategies for the identification of naïve and memory T-helper cells can also significantly influence the sensitivity of detection of CCR5 expression. Procedural methods are described that allow for the optimal measurement of naïve and memory T-helper cell CCR5 and CXCR4 expression as well as the quantitation of CCR5 and CXCR4 mRNA.