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Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II–induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy. Cancer Res; 73(4); 1328–39. ©2012 AACR.

PDF file - 490K, Figure S1: Cell line authentication data Figure S2: Full screen western blots for ChAT, VAChT, AChE and CHT1 Figure S3: Immunohistochemistry of ChAT and VAChT in normal human lung tissues Figure S4: Nicotine-induced ACh production in human BAC requires nAChRs, VAChT, and CHT1 Figure S5: VAChT and ChAT are expressed on human BACs Figure S6: Transfection of VAChT siRNA and Sigma-R siRNA suppress the expression of these proteins in human BACs Figure S7: Effect of VAChT siRNA on the apoptotic activity of vesamicol in nicotine-treated BACs Figure S8: Overexpression of pcDNA3-HA-Akt-CA in A549 and H358 human BAC cells

Capsaicin, the pungent ingredient of chili peppers, displays potent anti-neoplastic activity in a wide array of human cancer cells. The present manuscript examines the signaling pathways underlying the apoptotic activity of capsaicin in human small cell lung cancer (SCLC) in vitro and in vivo. Studies in neuronal cells show that capsaicin exerts its biological activity via the transient receptor potential vanilloid (TRPV) superfamily of cation-channel receptors. The TRPV family is comprised of six members (TRPV1-6). Capsaicin is a known agonist of the TRPV1 receptor. We observed that capsaicin-induced apoptosis in human SCLC cells was mediated via the TRPV receptor family; however it was independent of TRPV1. Surprisingly, the apoptotic activity of capsaicin required the TRPV6 receptor. Depletion of TRPV6 receptor by siRNA methodology abolished the apoptotic activity of capsaicin in SCLC cells. Immunostaining and ELISA showed that TRPV6 receptor was robustly expressed on human SCLC tissues (from patients) and SCLC cell lines but almost absent in normal lung tissues. This correlates with our results that capsaicin induced very little apoptosis in normal lung epithelial cells. The pro-apoptotic activity of capsaicin was mediated by the intracellular calcium and calpain pathway. The treatment of human SCLC cells with capsaicin increased the activity of calpain 1 and 2 by threefold relative to untreated SCLC cells. Such calpain activation, in response to capsaicin, was downstream of the TRPV6 receptor. Taken together, our data provide insights into the mechanism underlying the apoptotic activity of capsaicin in human SCLCs.

This chapter summarizes the variety of different assays available and their particular application in the field of infectious diseases. The discussion emphasizes general assay design, with important caveats relevant to test interpretation and development. The first immunoassays available measured milligram to microgram quantities of antibodies and relied primarily upon precipitation reactions between antigen and antibody. There are many classic agglutination assays used in the diagnosis of infectious diseases, such as the Widal test for typhoid fever. The spectrum of immunologic assays is discussed in detail. Enzyme immunoassays (EIAs) can be broadly classified as either homogeneous or heterogeneous assays. Noncompetitive indirect solid-phase ELISA is one of the most frequently employed immunoassays in a clinical laboratory. There are various types of anti-animal antibodies; the most commonly reported are human anti-mouse antibodies (HAMA). There are many different assay designs which combine an array of technologies, including chemiluminescence (CL), fluorescence immunoassay (FIA), flow cytometry, and molecular diagnostics (PCR and use of oligonucleotides and nanoparticles). As these assays provide rapid results and can be performed with very small sample sizes, they have wide applicability to epidemiological studies and vaccine trials. These assays are also especially suited to the assessment of multiple biological agents in a variety of samples.

By definition, immunoassays are biochemical assays that detect the presence of an analyte, either antibody or antigen, using labeled antibodies as the analytical reagent. Immunoassays can be adapted for detection of analytes across laboratory disciplines and are often more cost-effective than other diagnostic methods. In the clinical microbiology laboratory, immunoassays often serve as confirmatory tests, and therefore the results are typically not intended to be used as the sole basis on which a diagnosis is made. However, for certain infectious diseases, including Lyme disease, cryptococcal meningitis, and syphilis, antibody and antigen detection by immunoassays is the primary means by which the infection is established. Due to their ease of use, rapid turnaround time, and generally high specificity, immunoassays are increasingly becoming available for point-of-care testing. This chapter summarizes the common immunologic testing methods currently used in clinical microbiology laboratories and their application for the diagnosis of infectious diseases. The discussion emphasizes general assay design, with important caveats relevant to test development and interpretation. While some examples relating to clinical testing are included, for an in-depth, pathogen-focused discussion, the reader is directed to the designated chapters in this Manual.

BACKGROUND The ThinPrep® Pap Test™ (TP), a liquid-based cervical cytology preparation, was approved for use in the U.S. in 1996. The purpose of this study was to compare TP performance and biopsy follow-up studies with a similar population of high risk patients sampled by conventional Papanicolaou (Pap) smear (CS). METHODS Diagnostic and specimen adequacy interpretations for 2727 TP direct-to-vial Pap tests from a high risk university hospital practice were compared with 5000 CS preparations from the same physicians taken 1 year previously. Biopsy follow-up studies for the categories of squamous intraepithelial lesion (SIL), carcinoma, and atypical squamous cells of undetermined significance (ASCUS) for each time period and technique were contrasted. RESULTS The SIL/carcinoma detection rate increased from 7.7% to 10.5% (P < 0.01) and the ASCUS rate decreased from 12.5% to 6.9% (P < 0.01); the percentage of satisfactory but limited specimens decreased from 19.4% to 10.5% (P < 0.01). Low grade SIL cases increased by 57% (P < 0.01) whereas the 26% increase in high grade SIL cases was not statistically significant. Greater than 90% of ungraded SIL, high grade SIL, and carcinoma cases had abnormal biopsies by both the TP and CS methods. The number of biopsy-confirmed high grade dysplasias and carcinomas was similar in the two groups. A low grade SIL detected by TP was less likely to have an abnormal biopsy (70% vs. 85% for CS). Nevertheless, the 57% increase in low grade SIL diagnoses by TP resulted in more TP patients with dysplastic biopsy diagnoses. Follow-up studies for ASCUS cases diagnosed by either TP or CS were similar, and 21–24% of patients eventually were found to have dysplasia. CONCLUSIONS The TP technique appears to lead to the increased detection of low grade SIL lesions, decreased satisfactory but limited samples, and fewer equivocal specimens. No increase in biopsy-confirmed high grade dysplasias and carcinomas was found. Follow-up studies for the ASCUS category were nearly identical to those for CS. Cancer (Cancer Cytopathol) 1999;87:105–12. © 1999 American Cancer Society.

Background: Familial medullary thyroid cancer (MTC) is a form of type 2 multiple endocrine neoplasia in which individuals develop MTC as the sole phenotypic manifestation of their disease. A previous study has suggested that patients with familial MTC may have a later age of onset (and more indolent course) of MTC than is observed in individuals with multiple endocrine neoplasia type 2A. Methods and Results: The age-related penetrance of MTC, C-cell hyperplasia, and a positive pentagastrin test for carriers of a codon 609 mutation of the RET gene in a large MTC family was determined. Pentagastrin testing and surgical pathology findings for patients who had thyroidectomies were correlated with RET sequence analysis findings. The penetrance of this mutation for the development of MTC was 0% at age 20, 10% at age 30, 50% at age 45, and approximately 100% at age 60. The ages of onset of C-cell hyperplasia and a positive pentagastrin stimulation test were similar, and both preceded the age of onset of MTC. Carriers of the mutated gene in this family had a later age of onset of disease than has been reported for families with multiple endocrine neoplasia type 2A and 2B syndromes. Conclusions: These results may have implications for the clinical management of MTC families with a 609 mutation.

Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II–induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy. Cancer Res; 73(4); 1328–39. ©2012 AACR.

Pregnancies in women with systemic lupus erythematosus are recognized to result in excessive fetal morbidity and mortality. Maternal autoantibody status may explain some of these problems. Anti-cardiolipin antibody has been associated with recurrent pregnancy losses in some women with lupus, but the risk of these losses has not been defined. At the University of Pittsburgh between January 1, 1979, and December 31, 1989, an unmatched case-control study design was used to determine whether patients with lupus and anti-cardiolipin antibody (81 cases) were at increased risk for adverse pregnancy outcomes in comparison with lupus patients without the antibody (174 controls). Cases had 98 of 192 (51%) pregnancies with an adverse outcome, while controls had 212 of 494 (43%). The odds ratio for having any adverse pregnancy outcome was 1.40 (95% confidence interval (CI) 0.98-1.98). When pregnancies were classified according to specific adverse outcome types, the frequency of late miscarriages (14-20 weeks gestation) in cases and controls was 8% and 3%, respectively. The odds ratio for late miscarriage was 2.94 (95% CI 1.31-6.60). When pregnancies were stratified by birth number and by occurrence of pregnancy before or after diagnosis, the increased frequency of late miscarriages in cases was noted only in the first pregnancy when the pregnancy occurred before recognized disease. Preterm births (before 38 weeks gestation) were increased in cases compared with controls in pregnancies that occurred after diagnosis for second and third pregnancies. If a case had one previous adverse outcome, the odds ratio for another adverse outcome was 3.00 (95% CI 1.62-5.57). If a case had two previous adverse outcomes, the odds ratio for a third adverse pregnancy outcome was 4.14 (95% CI 1.62-10.58). Thus, a previous adverse pregnancy outcome was the most important risk factor for an adverse outcome in a subsequent pregnancy.

Background Small cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vitro. However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo. Methodology/Principal Findings BrdU assays and PCNA ELISAs showed that capsaicin displays robust anti-proliferative activity in four human SCLC cell lines. Furthermore, capsaicin potently suppressed the growth of H69 human SCLC tumors in vivo as ascertained by CAM assays and nude mice models. The second part of our study attempted to provide insight into molecular mechanisms underlying the anti-proliferative activity of capsaicin. We found that the anti-proliferative activity of capsaicin is correlated with a decrease in the expression of E2F-responsive proliferative genes like cyclin E, thymidylate synthase, cdc25A and cdc6, both at mRNA and protein levels. The transcription factor E2F4 mediated the anti-proliferative activity of capsaicin. Ablation of E2F4 levels by siRNA methodology suppressed capsaicin-induced G1 arrest. ChIP assays demonstrated that capsaicin caused the recruitment of E2F4 and p130 on E2F-responsive proliferative promoters, thereby inhibiting cell proliferation. Conclusions/Significance Our findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs.

A total of 57 schizophrenic patients (of which 17 were first-episode, neuroleptic naive) and 76 healthy controls were screened for anti-histone IgG antibodies using an enzyme immunoassay (ELISA). All patients had significantly higher anti-histone antibody titers than controls (t = 3.1, p less than 0.003). Previously medicated patients had significantly higher titers than neuroleptic-naive first episode patients (t = 2.87, p less than 0.006). This study suggests that neuroleptic medications are associated with anti-histone antibodies.

In the clinical setting, antidotes are generally administered after the occurrence of a drug overdose. Therefore, the most pertinent evaluation of any new agent should model human exposure. This study tested whether acetaminophen (APAP) hepatotoxicity was reversed when S-adenosyl-L-methionine (SAMe) was administered after APAP exposure, similar to what occurs in clinical situations. Comparisons were made for potency between SAMe and N-acetylcysteine (NAC), the current treatment for APAP toxicity. Male C57BL/6 mice were fasted overnight and divided into groups: control (VEH), SAMe treated (SAMe), APAP treated (APAP), N-acetylcysteine treated (NAC), SAMe or NAC administered 1 h after APAP (SAMe+APAP) and (NAC+APAP), respectively. Mice were injected Intraperitoneal (ip) with water (VEH) or 250 mg/kg APAP (15 ml/kg). One 1h later, mice were injected (ip) with 1.25 mmol/kg SAMe (SAMe+APAP) or NAC (NAC+APAP). Hepatotoxicity was evaluated 4 h after APAP or VEH treatment. APAP induced centrilobular necrosis, increased liver weight and alanine transaminase (ALT) levels, depressed total hepatic glutathione (GSH), increased protein carbonyls and 4-hydroxynonenal (4-HNE) adducted proteins. Treatment with SAMe 1 hr after APAP overdose (SAMe+APAP) was hepatoprotective and was comparable to NAC+APAP. Treatment with SAMe or NAC 1 h after APAP was sufficient to return total hepatic glutathione (GSH) to levels comparable to the VEH group. Western blot showed reversal of APAP mediated effects in the SAMe+APAP and NAC+APAP groups. In summary, SAMe was protective when given 1 h after APAP and was comparable to NAC.

Nutraceuticals are widely used by the general public, but very little information is available regarding the effects of nutritional agents on drug toxicity. Excessive doses of acetaminophen (APAP, 4-hydroxyacetanilide) induce hepatic centrilobular necrosis. The naturally occurring substance S-adenosyl-l-methionine (SAMe) has been reported to reduce the hepatic toxicity of APAP. The present study was designed to investigate the hepatoprotective effects of SAMe in comparison to the clinically used antidote N-acetylcysteine (NAC). Male C57BL/6 mice were injected intraperitoneally (i.p.) with an equimolar dose (1.25 mmol/kg) of either SAMe or NAC just before APAP, and the groups were denoted SAMe+APAP and NAC+APAP, respectively. Mice were immediately injected i.p. with 300 mg/kg APAP, and hepatotoxicity was evaluated after 4 h. SAMe was more hepatoprotective than NAC at a dose of 1.25 mmol/kg as liver weight was unchanged by APAP injection in the SAMe+APAP group, whereas liver weight was increased in the NAC+APAP group. SAMe was more hepatoprotective for APAP toxicity than NAC, because alanine aminotransferase levels were lower in the SAMe+APAP. Pretreatment with SAMe maintained total hepatic glutathione (GSH) levels higher than NAC pretreatment before APAP, although total hepatic GSH levels were lower in the SAMe+APAP and NAC+APAP groups than the vehicle control values. Oxidative stress was less extensive in the SAMe+APAP group compared with the APAP-treated mice as indicated by Western blots for protein carbonyls and 4-hydroxynonenal-adducted proteins. In summary, SAMe reduced APAP toxicity and was more potent than NAC in reducing APAP hepatotoxicity.

In a murine model of autoimmunity targeted against the epidermal cell Ags, Skn, adoptive transfer of Skn-immune T cells to immunosuppressed recipients elicits skin lesions in areas of mild epidermal trauma. In this study, we examined peripheral regulation of Skn-induced autoreactivity disrupted by rendering the mice immunoincompetent. We found that regulation of Skn-directed autoimmunity was restored by cotransfer of normal syngeneic spleen cells at twice the concentration of Skn-immune cells and was evidenced by significantly reduced lesion severity by days 5–7 post-cotransfer compared with animals given injections of Skn-immune cells alone. Enrichment and depletion of normal CD4+ or CD8+ spleen cells and RT-PCR analysis of selected cytokines identified CD4+ cells as the regulatory cells in the cotransfer inoculum; however, significant reduction in lesion severity was observed only when there was a concomitant increase in levels of IL-7. The role of IL-7 was further supported in that mice cotransferred with Skn-immune cells plus normal spleen cells, but also treated with anti-IL-7 Ab, no longer exhibited reduced lesion severity. To determine whether IL-7 expression without normal spleen cell cotransfer could modulate lesion development, an IL-7-encoding plasmid (pCMV-Tag1-IL-7) was topically delivered to sites flanking the stressed skin site in Skn-induced autoimmune mice. Daily application of 15 μg of pCMV-Tag1-IL-7 significantly suppressed lesion severity. Our results support a mechanism for CD4+ T cells and IL-7 in contributing to the control of autoreactivity.

Information is lacking regarding the biological response to environmental chemicals in the context of pre-existing disease. Benzo( α )pyrene (BaP), a polycyclic aromatic hydrocarbon, is a byproduct of combustion that causes renal injury and elicits a nephropathic response. This study evaluated the nephrotoxicity of BaP in normoglycemic and diabetic rats. Female Sprague Dawley rats were divided into four groups: normoglycemic-vehicle (NV), normoglycemic-BaP (N-BaP), diabetic-vehicle (DV) and diabetic-BaP (D-BaP). Diabetes was induced by intraperitoneal (ip) injection of streptozotocin (60 mg/kg, 1 ml/kg). Rats were injected (ip) with vehicle or 10 mg/kg BaP (1 ml/kg) once per week for 5 weeks. Urinary protein and albumin, plasma creatinine and light microscopy were performed to assess the effects of BaP on kidney function. Diabetes was confirmed by plasma glucose levels >400 mg/dl in the DV and D-BaP groups. BaP increased kidney weight and blood urea nitrogen (BUN) levels in the D-BaP relative to the DV group. No change in BUN was observed following 5 weeks of BaP treatment in the normoglycemic animals, however, kidney weight was increased ( p = 0.013) in the N-BaP relative to the NV animals. STZ diabetes increased susceptibility to BaP mediated renal damage following repeated treatment for 5 weeks when compared to age matched normoglycemic rats.

This study assessed the progression of renal damage in obese Zucker rats in response to deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Renal damage was evaluated by light microscopy and urine analysis at weekly intervals during the developmental phase of DOCA-salt hypertension and once during the plateau phase 42 days after the onset of treatment. Decreased tubular function was evident by day 8, as indicated by a significant increase in urine N-acetyl-beta-D-glucosaminidase activity and glucose excretion. The tubular index, a measure of tubular damage, was significantly elevated by day 15 and continued to increase throughout the experiment. Glomerular damage, which was evident by day 8, was followed by increased urine albumin excretion by day 15. Only a few sclerotic renal glomeruli were apparent before the plateau phase; however, by day 42, approximately 50% of the glomeruli were sclerotic. Hyperplastic vascular changes were mild at day 8 and slowly increased in severity during the developmental phase. By day 42 the vascular changes were severe with some vessels so hyperplastic that their lumens were almost occluded. These findings show progressive changes in renal structure and function that begin as early as day 8 and increase progressively until severe changes are present at day 42, resulting in an end-stage

Hepatic toxicity is associated with excessive dosages of the over the counter analgesic, acetaminophen (APAP). The aim of this study was to explore protection by the nutritional agent S-adenosylmethionine (SAMe) on APAP hepatotoxicity. Male C57BL/6 mice were injected intraperitoneal (i.p.) with 500 mg/kg (15 ml/kg) APAP or water vehicle (VEH). SAMe was injected i.p. at a dose of either 1000 mg/kg (5 ml/kg) just prior or 500 mg/kg SAMe 15 min prior to administration of VEH or APAP. Comparison of groups showed that SAMe reduced APAP toxicity. Plasma alanine aminotransferase (ALT) levels were increased 2 and 4 h after APAP administration when compared to vehicle (VEH) controls. Liver weight was increased relative to the VEH group within 4 h after APAP treatment. Histological examination by light microscopy confirmed small changes in morphology within 2 h after APAP injection and marked centrilobular necrosis within 4 h in the APAP group. In contrast, when APAP was administered to SAMe pretreated mice, ALT and liver weights were comparable to the VEH and SAMe groups. Histological examination also showed that SAMe produced a marked protection in APAP mediated centrilobular necrosis at 4 h after APAP injection. APAP administration depressed hepatic glutathione levels when monitored at 2 and 4 h. Lipid peroxidation was induced above VEH values 2 and 4 h after APAP injection. Consistent with the SAMe protection of APAP hepatic toxicity, the expected depletion of hepatic glutathione (GSH) levels by APAP was prevented by SAMe pretreatment. SAMe pretreatment also prevented the induction of lipid peroxidation at 2 and 4 h post-APAP administration. In conclusion, SAMe provides protection from APAP hepatic toxicity at 2 and 4 h post-APAP injection. SAMe pretreatment prevented APAP associated depletion in hepatic glutathione and induction of lipid peroxidation as part of its mechanism of protection.

The Association of Medical Laboratory Immunologists (AMLI) have developed a panel of antinuclear and anticytoplasmic antibody consensus sera that can be useful for enzyme immunoassay (EIA), Ouchterlony, and immunofluorescence assay methods. It was developed to assist in the evaluation of newly available EIA methods for the detection of autoantibodies. The panel of sera was evaluated in several clinical laboratories and a large number of laboratories owned by manufacturers of clinical autoantibody testing kits. The majority of sera performed well for the EIAs in both the clinical laboratories and the manufacturers' laboratories, but some samples had discrepant results. A major source of discrepancy is the current inability of the EIA results to be directly compared in a quantitative way as no standardization exists. The evaluation demonstrated lower sensitivity of detection by the Ouchterlony method. The limited evaluation of the sera with immunoblotting and Western blotting did not show good agreement with other methods. Further work must be done to standardize blotting methods prior to their use in routine clinical testing. The sera are now available to vendors and clinical laboratories for use in the detection of SS-A, SS-B, Sm, U1-RNP, Scl-70, Jo-1, double-stranded DNA, and centromere antibodies. The availability of the consensus sera will help evaluate and improve the EIA methods currently being used.

Infection with varicella-zoster virus (VZV) is common, occurring as primary varicella, usually during childhood, and as zoster, following reactivation of latent virus. Although serious VZV infections are infrequent, they can be life-threatening when they occur in susceptible immunosuppressed patients (1, 2). These patients have a high incidence of visceral involvement, including pneumonitis, meningoencephalitis, and rarely, hepatitis. We report here 3 cases of VZV hepatitis in adult liver transplant recipients that occurred in our institution between 1984 and 1989. We describe their clinical presentations and use these cases to illustrate some aspects in diagnosis and prevention of VZV infection after solid organ Tx.