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The synthesis and characterisation of a novel Pt(IV) monocarboxy precursor complex c,c,t-[Pt(DACH)(Ox)(OEt)(Suc)], which can be employed to synthesise mono functionalised Pt(IV) oxaliplatin-based complexes is reported. Reaction of c,c,t-[Pt(DACH)(Ox)(OEt)(Suc)] with a tumour penetrating peptide, TPP, afforded the corresponding novel monofunctionalised Pt(IV) peptide conjugate c,c,t-[Pt(DACH)(Ox)(OEt)(SucTPP)] in high purity. Conjugation of TPP to c,c,t-[Pt(DACH)(Ox)(OEt)(Suc)] was demonstrated to ameliorate its inherent cytotoxicity fifteen fold in the Pt sensitive HCT116 colorectal cancer cell line and two fold in the relatively resistant HT29 colorectal cancer cell line.

Novel Pt(iv) tumour penetrating peptide (TPP) conjugates are reported. They are the first example of metallodrugs to target a membrane bound heat shock protein 70 positive (memHSP70+) phenotype in cancer cells. The conjugates exhibit superior cytotoxicity as compared to oxaliplatin alone in Pt resistant colorectal cancer cells with relatively high memHSP70+ expression. Substitution of TPP in Pt(iv) peptide conjugates with scrambled peptide (ScP) essentially abolishes the observed cytotoxicity.

We describe a novel improved approach to the synthesis of the important and well-known heat shock protein 70 inhibitor (HSP70), pifithrin-μ, with corresponding and previously unreported characterisation. The first example of a combination study comprising HSP70 inhibitor pifithrin-μ and cisplatin or oxaliplatin is reported. We have determined, using the Chou-Talalay method, (i) moderate synergistic and synergistic effects in co-treating PC-3 prostate cancer cells with pifithrin-μ and cisplatin and (ii) significant synergistic effects including strong synergism in cotreating HT29 colorectal cancer cells with oxaliplatin and pifithrin-μ.

We describe a novel improved approach to the synthesis of the important and well-known heat shock protein 70 inhibitor (HSP70), pifithrin-μ, with corresponding and previously unreported characterisation. The first example of a combination study comprising HSP70 inhibitor pifithrin-μ and cisplatin or oxaliplatin is reported. We have determined, using the Chou-Talalay method, (i) moderate synergistic and synergistic effects in co-treating PC-3 prostate cancer cells with pifithrin-μ and cisplatin and (ii) significant synergistic effects including strong synergism in cotreating HT29 colorectal cancer cells with oxaliplatin and pifithrin-μ.

SummarySecondary bacterial pneumonia frequently claimed the lives of victims during the devastating 1918 influenza A virus pandemic. Little is known about the viral factors contributing to the lethality of the 1918 pandemic. Here we show that expression of the viral accessory protein PB1-F2 enhances inflammation during primary viral infection of mice and increases both the frequency and severity of secondary bacterial pneumonia. The priming effect of PB1-F2 on bacterial pneumonia could be recapitulated in mice by intranasal delivery of a synthetic peptide derived from the C-terminal portion of the PB1-F2. Relative to its isogenic parent, an influenza virus engineered to express a PB1-F2 with coding changes matching the 1918 pandemic strain was more virulent in mice, induced more pulmonary immunopathology, and led to more severe secondary bacterial pneumonia. These findings help explain both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic.

Melatonin has gained recent popularity as a treatment for insomnia and other sleep disorders; however, its cellular effects are unknown. We report the effects of melatonin on the cellular morphology of Chinese hamster ovary (CHO) cells transformed to express the human melatonin receptors, mt1 and MT2. Our results show that melatonin exerts a strong influence on cellular shape and cytoskeletal organization in a receptor-dependent and possibly subtype-selective manner. The cell shape change that we see after a 5-h treatment of these non-neuronal cells with a pharmacological concentration of melatonin consists of the formation of long filamentous outgrowths that are reminiscent of the neurite processes produced by differentiating nerve cells. This morphological change occurs exclusively in cells expressing the mt1 receptor. We find that the microtubule and microfilament organization within these outgrowths is similar to that of neurites. Microtubules are required for the shape change to occur as Colcemid added in combination with melatonin completely blocks outgrowth formation. We demonstrate that the number of cells showing the altered cell shape is dependent on melatonin concentration, constant exposure to melatonin and that outgrowth frequencies increase when protein kinase A (PKA) is inhibited. Concomitant melatonin-dependent increases in MEK 1/2 and ERK 1/2 phosphorylation are noted in mt1-CHO cells only. The production of filamentous outgrowths is dependent on the translation of new protein but not the transcription of new mRNA. Outgrowth number is not controlled by centrosomes but is instead controlled by the polymerization state of the actin cytoskeleton. The results of this work show that the organization of the cytoskeleton is affected by processes specifically mediated or regulated by the mt1 receptor and may represent a novel alternative mechanism for the stimulation of process formation.

More than 250 coliform and noncoliform bacteria, isolated by standard membrane filtration methods from rural, untreated groundwater supplies, were examined for resistance to 16 antibiotics. All of the noncoliforms and 87% of the coliforms were resistant to at least one antibiotic, with resistance most commonly directed toward novobiocin, cephalothin, and ampicillin. The frequency of multiple antibiotic resistance (MAR) within each species was also determined. Approximately 60% of the coliforms were MAR, including 14, 64, and 94% of Escherichia coli, Citrobacter freundii, and Enterobacter cloacae isolates, respectively. In comparison, MAR was demonstrated by more than 95% of the noncoliforms and included isolates of Acinetobacter calcoaceticus, Aeromonas hydrophila, and Serratia marcescens. Ampicillin-resistant environmental isolates were assayed for ability to transfer resistance to ampicillin-sensitive strains of E. coli and Salmonella typhimurium. Environmental E. coli isolates were capable of in vitro transfer to both recipients in a nutrient-rich environment (trypticase soy broth) at frequencies ranging from 1.1 × 10−5 to 1.0 × 10−4. Isolates of C. freundii and Klebsiella pneumoniae exhibited resistance transfer to the E. coli recipient under similar conditions at reduced rates. None of the environmental isolates demonstrated transfer of ampicillin resistance when placed in an environment containing filter-sterilized well water. The presence of antibiotic resistant bacteria, and particularly MAR bacteria, in rural groundwater supplies used as drinking water sources may have important public health implications.

Feeding difficulties have been identified as one of the main factors that contribute to weight loss and poor nutritional status in people with dementia. Previous research has looked into the behaviours that patients with dementia exhibit when eating. It is not known what behavioural problems at mealtimes are the most common or how these behaviours relate to nutritional status among patients with dementia. This study aims to investigate the frequency of feeding difficulties seen among elderly patients with dementia in a primary care setting in the Republic of Ireland in order to inform resources for patients and carers and community dietitians. Eighty-seven out of one hundred eligible patients who met the study criteria (i.e. greater than 65 years old with a diagnosis of dementia) consented to take part in the study. Nutritional risk was measured using both the Malnutrition Universal Screening Tool (MUST) and the Mini Nutrition Assessment (MNA). The Edinburg feeding and evaluation questionnaire (ED FED-Q) was used to categorise and measure behavioural problems at mealtimes. All tools were carried out by the same community dietitian (MMK).

To the Editor, We read with interest the recent Cytojournal article “Collection of the BD SurePath Pap Test with a broom device plus endocervical brush improves disease detection when compared to the broom device alone or the spatula plus endocervical brush combination” by Davis-Devine et al.,[1] in which the authors conclude that a combination of broom and brush is superior to broom alone as collection devices for Surepath® Pap tests, increasing the endocervical cell recovery and the yield of low-grade squamous lesions (LSIL). We wish to report our findings from a similar study we conducted as they are somewhat different from those of Davis-Devine et al.[1] and may be of interest to the readers of Cytojournal. Preliminary findings from our study were presented at the United States and Canadian Academy of Pathology 2008 Annual Meeting.[2] In our study, we specifically attempted to answer the question of whether the endocervical (EC) brush (Rovers® EndoCervex-Brush®) (“brush”) demonstrated any additional utility when used in combination with the Rovers® Cervex® brush (“broom”), with respect to endocervical cell recovery or diagnostic rate in BD Surepath® Pap tests. A total of 14,147 BD Surepath® vials were visually inspected in two randomly selected study periods (April 27, 2007 to July 2, 2007 and September 14, 2007 to December 28, 2007) and the presence and type of sampling devices were recorded. A total of 12,089 vials contained the broom alone, while 1,897 had a broom + brush combination. In addition, 125 vials contained only a brush and 36 contained no sampling device. We then correlated the presence of a broom only or broom + brush combination with patient age, rate of EC cell recovery, and diagnostic rate [Table 1] and rate of each particular Bethesda 2001 diagnostic category [Table 2]. Table 1 Characteristics of broom versus broom + brush groups Table 2 Rate of each Bethesda 2001 diagnostic category We found that the broom + brush combination was used to sample older women, which may indicate that clinicians who submit specimens to our laboratory may be preferentially using the EC brush to ensure sampling of the transformation zone (TZ) in older women. Despite the fact that it was used in older women, the broom + brush combination ensured a higher rate of EC cell recovery than the broom alone. We found no significant differences in the overall detection rate of abnormalities or for any diagnostic category between the Pap tests collected with the broom + brush combination and those collected with the broom only. However, since women sampled with the broom + brush combination were significantly older than those sampled with the broom alone, we had expected a lower abnormal rate in the broom + brush combination group based on the documented decline in cervical abnormalities with increasing age. In trying to elucidate the reasons why we did not find a higher abnormal or a higher LSIL detection rate while Davis-Devine et al.[1] did, we considered two possible explanations. Our study was slightly smaller in size with 12,089 broom and 1,897 broom + brush cases, as compared to 11,130 broom and 2,921 broom + brush cases in their study, but we believe that this difference did not significantly impact our respective results. It is more likely that the differences in the results between the two studies are related to differences in the two patient populations undergoing screening. The population in the study by Davis-Devine et al.[1] had a higher calculated abnormal rate (10.5% vs. 7.4% in our study), largely due their higher rates of ASC-US (6.4% vs. 4.3%) and LSIL (3.1% vs. 2%), while their HSIL rate was lower (0.23% vs. 0.31%). Abnormal Pap test rates vary considerably in the literature, likely reflecting both the potential variability in cytologic diagnoses between institutions and a combination of different patient population characteristics such as age, location, socioeconomic status, prevalence of HPV, and HIV infection as well as initial and continued access to adequate preventive healthcare. Davis-Devine et al.[1] do not provide age data for their population, however, based on the higher rate of both ASC-US and LSIL we suspect that their study population may be younger than ours. Like Davis-Devine et al., our study was probably underpowered for comparisons in detection rates of HSIL and more severe diagnoses. In conclusion, the results of our study support the idea that a better sampling of the EC/TZ can be achieved with the broom + brush combination as compared to sampling with the broom alone. However, we believe that additional larger studies on the subject of collection devices, including the newer and potentially easier to use Rovers® Cervex-Brush® Combi (which combines the broom and the brush into one device) in the setting of liquid-based Pap testing are needed. Such studies need to allow age-controlled comparisons and correlation with human papillomavirus DNA test status before we can conclude that sampling with any device or combination of devices allows the detection of a higher rate of significant cervical abnormalities.

BACKGROUND Apart from several subjective criteria, ASCUS and LSIL are defined by nuclear enlargement of 2.5× to 3× and ≥3× the area of a normal intermediate squamous cell nucleus, respectively. The aim of this study was to assess the ability of observers with various degrees of experience to estimate nuclear area ratios. METHODS. Forty-five participants (5 anatomic pathologists, 5 cytopathologists, 2 cytopathology fellows, 16 pathology residents, 8 cytotechnologists, 6 medical students, and 3 cytopreparatory staff members) judged the area ratios of pairs of squamous cell nuclei imaged at ×100 objective magnification. For Test 1 (T1), participants chose between 5 preset area ratios (1.0× to 1.4×, 1.5× to 1.9×, 2.0× to 2.4×, 2.5× to 2.9×, 3.0× to 3.4×) for 15 pairs of cells with ratios falling in the middle of these intervals. One week after T1, an instructional tutorial was given and T1 was repeated in different order of cell pairs as Test 2 (T2). RESULTS. The kappa values for all participants were 0.30 for T1 and 0.39 for T2. Accurate responses were given in 50.5% in T1 and 53.5% in T2. Both T1 and T2 responses failed to differentiate between area ratios corresponding to ASCUS and LSIL. There were no differences in accuracy according to sex, experience, or expertise in cytopathology. CONCLUSIONS. The overall accuracy of participants in estimating nuclear area ratios was low, especially for ratios in the range of ASCUS versus LSIL, with only minimal improvement after an instructional tutorial. Nuclear area ratio estimation may, therefore, contribute to the well-known substantial variability in ASCUS rates and ASC/squamous intraepithelial lesion ratios. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.

BACKGROUND. The Bethesda 2001 (B2001) classification system raised the threshold for reporting atypical squamous cells. The category of Atypical Squamous Cells of Undetermined Significance (ASCUS)-favor reactive has been eliminated with a recommendation for cytologists to judiciously downgrade those Papanicolaou (Pap) tests that would formerly have been in this category to Negative for Intraepithelial Lesion or Malignancy (NILM). The effect of this change on sensitivity and specificity of the Pap test is not yet known. METHODS. A total of 535 consecutive SurePath Pap tests interpreted as ASCUS during a period from March 1 through December 31, 2001, with follow-up polymerase chain reaction(PCR)-based human papilloma virus (HPV) testing, were independently reviewed by 3 pathologists and 1 cytotechnologist who reinterpreted these tests according to Bethesda 2001 criteria. Follow-up HPV and biopsy results were compiled for a 5-year period. RESULTS. By consensus of the 4 observers, 169 (32%) of the ASCUS cases were downgraded to NILM. These cases showed a lower rate of underlying high-risk HPV infection (11% vs 30%) and cervical intraepithelial neoplasia of grades 2 to 3 (CIN 2/3) (5% vs 10%) on follow-up than those tests that were reinterpreted as ASCUS or higher. Nine women with follow-up CIN 2/3 would have had the Pap test interpreted as NILM instead of ASCUS under Bethesda 2001 (20% of all CIN 2/3 found). Individual reviewers downgraded 29% to 42% Pap tests to NILM including those of 5 to 10 women with follow-up CIN 2/3. CONCLUSIONS. The ASCUS threshold established by B2001 prevents a sizeable subset of women from having follow-up for ASC. However, as a consequence, a few women with CIN 2/3 are downgraded to NILM. The cost savings thus achieved must be weighed against the lost opportunities to detect CIN 2/3. Cancer (Cancer Cytopathol) 2008. © 2008 American Cancer Society.

Thymic carcinoid tumors (well-differentiated neuroendocrine carcinomas) are uncommon anterior mediastinal neoplasms. These tumors are frequently accompanied by other endocrinopathies as part of a multiple endocrine neoplasia type I syndrome (MEN type I) and by paraneoplastic Cushing's syndrome and have a poor prognosis. We present the case of a 24-year-old man who presented for follow-up of thymic carcinoid with extensive bony metastases. He had recently completed radiotherapy to lesions involving his skull and mandible. An ultrasound-guided left-sided diagnostic and therapeutic thoracentesis was performed yielding 1 l of cloudy yellow fluid. The cytologic fluid preparations consisted of large “cannonballs” and atypical cell groups with salt and pepper nuclear chromatin. A panel of immunohistochemical stains were performed on the cell block material, and the atypical cells were positive for cytokeratin, synaptophysin, and chromogranin, but not for TTF1. These findings were consistent with metastatic well-differentiated neuroendocrine carcinoma (carcinoid tumor). This is the first reported case of a carcinoid tumor manifesting as large, spherical, smoothly contoured cell aggregates (“cannonballs”) in a pleural fluid. Despite its rarity, a metastatic carcinoid tumor should be considered when “cannonballs” are found in effusions. Diagn. Cytopathol. 2008;36:333–337. © 2008 Wiley-Liss, Inc.

Melatonin induces cellular differentiation in numerous cell types. Data show that multiple mechanisms are involved in these processes that are cell-type specific and may be receptor dependent or independent. The focus of this study was to specifically assess the role of human MT1 melatonin receptors in cellular differentiation using an MT1-Chinese hamster ovary (CHO) model; one that reproducibly produces measurable morphologic changes in response to melatonin. Using multiple approaches, we show that melatonin induces MT1-CHO cells to hyperelongate through a MEK 1/2, and ERK 1/2-dependent mechanism that is dependent upon MT1 receptor internalization, Gi protein activation, and clathrin-mediated endocytosis. Using immunoprecipitation analysis, we show that MT1 receptors form complexes with Gi(alpha) 2,3, Gq(alpha), beta-arrestin-2, MEK 1/2, and ERK 1/2 in the presence of melatonin. We also show that MEK and ERK activity that is induced by melatonin is dependent on Gi protein activation, clathrin-mediated endocytosis and is modulated by microtubules. We conclude from these studies that melatonin-induced internalization of human MT1 melatonin receptors in CHO cells is responsible for activating both MEK 1/2 and ERK 1/2 to drive these morphologic changes. These events, as mediated by melatonin, require Gi protein activation and endocytosis mediated through clathrin, to form MT1 receptor complexes with beta-arrestin-2/MEK 1/2 and ERK 1/2. The MT1-CHO model is invaluable to mapping out signaling cascades as mediated through MT1 receptors especially because it separates out MEK/ERK 1/2 activation by MT1 receptors from that of receptor tyrosine kinases.

A review of our institution's Papanicolaou test records over an 11-yr period showed that liquid-based Papanicolaou tests (LBPTs) had a significantly higher frequency of diagnoses of Herpes simplex virus (HSV)-related cellular changes compared to conventional Papanicolaou smears (77/302,841, 0.026% vs. 56/376,173, 0.015%, P = 0.002). To investigate the accuracy of the diagnosis of HSV by LBPT, we performed conventional polymerase chain reaction (PCR) on the residual samples from 258 prospectively collected LBPT and real-time PCR using a different primer set on a subset of 40 LBPT. Conventional PCR was positive in 22 of 22 cases diagnosed of HSV, 1 of 2 cases diagnosed as suspicious for HSV, and none of 234 LBPT without a cytologic HSV diagnosis. Real-time PCR was positive in 8 of 8 cases diagnosed as HSV and none of the 32 controls. We conclude that LBPT allows an increased detection of HSV that is highly accurate. Diagn. Cytopathol. 2008;36:94–103. © 2008 Wiley-Liss, Inc.

Pneumonia caused by Streptococcus pneumoniae is a significant cause of morbidity and mortality during influenza virus epidemics. We had previously advanced the hypothesis that interactions of pneumococcus with the receptor for platelet activating factor (PAFR) in the lung were facilitated by antecedent influenza virus infection and play a major role in the pathogenesis of bacterial super-infections. Although influenza enhanced the adherence of pneumococci to respiratory epithelial cells in vitro, chemical or antibody-mediated blockade of the PAFR did not affectt adherence. In agreement with these data, mice lacking PAFR had similar bacterial loads within the lung compartment when compared to heterozygous littermates and were not protected from secondary pneumococcal pneumonia after influenza. Lack of support for this hypothesis and the observation of enhanced inflammation during secondary pneumococcal pneumonia in mice lacking PAFR may moderate enthusiasm for treatment strategies targeting the interaction of bacteria with PAFR.

We investigated the morphological features of Trichomonas vaginalis in liquid-based Papanicolaou (Pap) (LBP) tests, in order to assess the impact of this new slide preparation system on recognition of T. vaginalis organisms. We reviewed 88 LBP test slides previously interpreted as showing T. vaginalis and assessed morphological characteristics of the organisms and the presence of secondary features. Eighty-six of 88 slides showed, at least focally, organisms with cytoplasmic granules, nuclei, and flagella allowing for confident recognition of T. vaginalis. Compared with 58 controls, cases of T. vaginalis were more likely to have squamous cells with perinuclear halos, “ghost cells,” and coccobacilli. The lack of a “dirty” background or cytolysis allowed a more straightforward identification of the organisms. We concluded that T. vaginalis organisms are morphologically distinct in LBP tests, which may lead to an increased specificity when they are identified in such tests. Diagn. Cytopathol. 2005;32:253–259. © 2005 Wiley-Liss, Inc.

No standardized PCR method is available for the laboratory diagnosis of the pertussis syndrome. Consensus recommendations for the use of PCR in the diagnosis of Bordetella pertussis infections have been proposed, and the aim of this study was to develop a method that fulfills all of these criteria. A rapid-cycle shared-primer PCR method with a microwell format and probe hybridization detection step (POR) was developed using novel oligonucleotides targeted to the outer membrane porin gene ( Bordetella spp.). In specimens positive for Bordetella spp., B. pertussis was differentiated from Bordetella parapertussis and Bordetella bronchiseptica by hybridization with organism-specific oligonucleotide probes. An internal control was developed using overlap extension PCR and mouse β-actin DNA. The analytical specificity was 100%. The analytical sensitivity was comparable to that of nested IS 481 and IS 1001 PCR (∼1 organism per reaction). The clinical sensitivity and specificity were ascertained using 705 specimens (from 705 patients). The results were compared to those of a nested-PCR method targeting the insertion sequences IS 481 and IS 1001 . Fifty-one specimens were positive for B. pertussis by POR and IS 481 PCR. Two specimens which fulfilled a clinical definition of pertussis were positive by POR and negative by IS 481 PCR. A total of 652 specimens were negative by both methods. B. parapertussis was not detected in any specimens. PCR inhibition was detected in 21 out of 705 specimens (2.98%). Thus, a rapid (4 h, including specimen preparation) PCR method which fulfills all of the consensus recommendations was developed and validated for the detection of B. pertussis .