Protein phosphorylation and tyrosine kinase activity in medullary thyroid carcinomas of the rat

The kinetics of endogenous protein phosphorylation and resultant phosphoprotein patterns were investigated in well-differentiated (DMTC) and undifferentiated (AMTC) medullary thyroid carcinomas of the rat. Cytosolic or particulate fractions from these tumors were incubated with gamma-32P-ATP in the presence of various effectors. Phosphorylation appeared to be predominantly independent of exogenously added cyclic AMP. Magnesium and manganese were equally effective cofactors. For both tumor types 32P incorporation into cytosolic proteins was maximal within 3-4 min after addition of ATP and subsequently decreased gradually within 1 h. In contrast, in particulate preparations maximal incorporation was reached within 30 s and remained constant over a relatively long time span. In both cases, however, 32P incorporation in extracts from DMTCs were 50-100% higher as compared to AMTCs. Comparison of the phosphoprotein patterns of each tumor after in vitro phosphorylation showed some significant differences. A phosphoprotein with molecular weight of 90 kilodalton (90 kD) was exclusively expressed in the cytosol of DMTC, whereas 99- and 84-kD phosphoproteins were only present in the cytosol of AMTC. The DMTC particulate fraction contained two phosphoproteins (with molecular weights of 40 and 37 kD), which were absent from that of AMTC. In addition, a number of proteins were more intensely phosphorylated in one of the tumors, e.g. proteins of 94 and 33 kD in DMTC cytosol and a protein of 78 kD in AMTC cytosol. Calcium induced phosphorylation of five proteins in DMTC cytosol (with molecular weights of 69, 55, 49, 43 and 32 kD), which were less intensely or not phosphorylated in AMTC. Tyrosine kinase activity was investigated using exogenously added poly(glutamine:tyrosine, 4:1) as an artificial substrate. Cytosolic tyrosine kinase activity in DMTC was +/- 50% higher than in AMTC (11.9 +/- 0.6 and 8.2 +/- 1.7 pmol/mg/min, respectively). The enzyme activities in the particulate preparations were much higher than in the cytosols (+/- 100 pmol/mg/min), although considerable variations in enzyme activity between different tumors of either type were observed. Quantitative differences in tyrosine kinase activity between AMTC and DMTC particulate fractions did not seem to exist using this substrate. Phosphoamino acid analysis of endogenously phosphorylated proteins in both AMTC and DMTC showed phosphotyrosine to be present only in cytosolic proteins within the 50-kD molecular weight region, the majority of 32P being on serine and some on threonine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic,household and spatial clustering of leprosy on an island in Indonesia: a population-based study

Background  

It is generally accepted that genetic factors play a role in susceptibility to both leprosy per se and leprosy type, but only few studies have tempted to quantify this. Estimating the contribution of genetic factors to clustering of leprosy within families is difficult since these persons often share the same environment. The first aim of this study was to test which correlation structure (genetic, household or spatial) gives the best explanation for the distribution of leprosy patients and seropositive persons and second to quantify the role of genetic factors in the occurrence of leprosy and seropositivity.     

Comparison of PCR with the routine procedure for diagnosis of tuberculosis in a population with high prevalences of tuberculosis and human immunodeficiency virus

Direct smear examination with Ziehl-Neelsen (ZN) staining for the diagnosis of tuberculosis (TB) as employed in most low-income countries is cheap and easy to use, but its low sensitivity is a major drawback. The low specificity of chest X-rays, used for the diagnosis of smear-negative TB, risks high levels of overdiagnosis. Major advances in molecular techniques, which rapidly identify mycobacterial DNA in sputa, may overcome these obstacles. In this study, the AMPLICOR PCR system was used to diagnose pulmonary TB in a developing country with high prevalences of both TB and human immunodeficiency virus (HIV). The sensitivity and specificity of this technique were compared to those of the usual diagnostic techniques. Sputum specimens were collected from 1,396 TB suspects attending the Rhodes Chest Clinic, Nairobi, Kenya. The specimens were analyzed for the presence of Mycobacterium tuberculosis by PCR; culture on Löwenstein-Jensen medium was used as the “gold standard.” All culture-positive samples were genotyped to identify the mycobacterial species. The sensitivity and specificity of PCR were 93 and 84%, respectively. HIV status did not affect the sensitivity of PCR. A total of 99.7% of the true smear-positive and 82.1% of the true smear-negative TB patients were correctly identified by PCR. PCR detected M. tuberculosis in 11.7% of the culture-negative suspects, 60% of which had one or two PCR-positive sputum specimens. Of the 490 positive cultures, 486 were identified as M. tuberculosis. The high sensitivity of Amplicor PCR merits usage in a clinical setting with high TB and HIV burdens. Thus, PCR can be considered as an alternative to ZN staining in combination with chest X-ray for diagnosis of TB; however, cost-effectiveness studies and operational studies are required to support an evidence-based decision of introducing PCR for TB control in high-burden environments.Tuberculosis (TB) is one of the most serious infectious diseases and a considerable public health problem due to its high risk of person-to-person transmission, morbidity, and mortality. Both the human immunodeficiency virus (HIV) epidemic and social deterioration have contributed to the overall increase in the Mycobacterium tuberculosis infection rate, especially in developing countries, where resources are scarce (13). In Nairobi the case detection rate increased from 78 per 100,000 in 1991 to 581 per 100,000 in 2001, with a total number of 12,963 cases.Early diagnosis followed by adequate treatment is essential to prevent both morbidity and mortality. Although the conventional technique of direct smear examination with Ziehl-Neelsen staining (ZN) is cheap and easy to perform, its low sensitivity is a major drawback. Depending on the number of specimens examined, ZN detects 30 to 60% of the culture-positive “TB suspects” (7). Furthermore, it requires sputum samples collected on consecutive days, making the procedure slow and making patient compliance with the diagnostic process difficult.New techniques are very much needed (7), and molecular amplification assays such as PCR have been shown to be promising alternatives even for developing countries (2). PCR has the potential to be a cost-effective alternative, provided the diagnosis can be determined with one sputum examination (8). If diagnosis can be established faster, and the diagnostic process becomes less cumbersome for the patient, PCR may reduce delay both in diagnosis and in the start of treatment.Depending on the “gold standard” and other methodological factors, studies show PCR sensitivities ranging from 77% to more than 95% and PCR specificities of >95% for smear-positive specimens (4, 9, 10, 12). However, sensitivities for smear-negative TB patients have been reported to be below 90% (9). Most PCR studies have been performed in industrial countries (4, 9, 10, 12) where the TB and HIV burdens are low.To investigate the performance and feasibility of PCR in an environment of TB endemicity and high prevalences of HIV and AIDS, a study was conducted in Nairobi, Kenya, comparing PCR to conventional routine diagnostic methods within a program setup. In this study, the Roche Amplicor Mycobacteria PCR test for the direct detection of M. tuberculosis was used on sputum specimens from TB suspects attending a chest clinic in Nairobi. Its performance was compared with those of the basic routine diagnostic procedures according to the national guidelines (6), including clinical findings, ZN, and chest X-rays (CXR), on smear-negative suspects. Löwenstein-Jensen (LJ) culture results were used as the gold standard.     

Use of PCR for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis

Microscopy and PCR were compared for use in the diagnosis of post-kala-azar dermal leishmaniasis (PKDL) in 63 patients. Aspirates of lymph nodes (samples from 52 patients), skin (23 samples), and bone marrow (18 samples) were used. For 11 patients lymph node aspiration could be repeated 6 months after they recovered from PKDL. During active PKDL, PCR was positive for 42 of 52 (80.8%) lymph node aspirates and 19 of 23 (82.7%) skin aspirates, whereas microscopy was positive for only 9 of 52 (17.3%) lymph node aspirates and 7 of 23 (30.4%) skin aspirates. PCR was always positive when parasites were seen by microscopy. When the results obtained with lymph node and skin aspirates from the same patient (n = 16) were compared, there was complete agreement. Bone marrow samples were negative by microscopy and PCR for 16 patients and positive by both methods for 1 patient; for one sample only the PCR was positive. PCR confirmed the co-occurrence of visceral leishmaniasis and PKDL in one patient and confirmed the suspicion of this co-occurrence in the other patient. After recovery, no parasites were found by microscopy, but 2 of 11 (18.2%) samples were still positive by PCR. Thirty negative controls were all found to be PCR negative, and 15 positive controls were all PCR positive. Cross-reactions with Mycobacterium leprae could be ruled out. In conclusion, PCR with inguinal lymph node or skin aspirates is suitable for confirming the clinical diagnosis of PKDL. In some patients, lymph node aspirates are probably preferred because aspiration of material from the skin may leave scars.     

Evaluation of PCR for diagnosis of visceral leishmaniasis.

An evaluation of Leishmania PCR was performed with bone marrow, lymph node, and blood samples from 492 patients, 60 positive controls, and 90 negative controls. Results were compared with microscopy results for Giemsa-stained smears. PCR and microscopy of lymph node and bone marrow aspirates from patients with microscopically confirmed visceral leishmaniasis (VL) were equally sensitive. However, in patients clinically suspected of having VL and in whom parasites could not be demonstrated by microscopy, PCR was positive for 12 of 23 (52.2%) lymph node aspirates and 8 of 12 (66.7%) bone marrow aspirates, thus confirming the clinical diagnosis of VL. With PCR on filter paper, Leishmania DNA was detected in the blood of 33 of 47 (70%) patients with confirmed VL and in 2 of 11 (19%) patients suspected of having VL. Positive PCR results were more frequently found for blood samples on filter paper than for samples stored in EDTA. In conclusion, PCR is a more sensitive method than microscopy for the detection of Leishmania in lymph node and bone marrow aspirates, being especially useful for the confirmation of cases of suspected VL. Blood from a finger prick may be used for the initial PCR screening of people suspected of having VL. If the PCR of blood is negative, one should perform PCR with lymph node and/or bone marrow material, because PCR with these materials is more often positive.     

Clinical heterogeneity of familial colorectal cancer and its influence on screening protocols.

The age at onset of non-polyposis colorectal cancer (CRC) was investigated in 49 families with at least three relatives affected in two successive generations. Forty one of these families satisfy the accepted minimum criteria for hereditary non-polyposis CRC. The remaining eight families were distinguished by a late age of disease onset and, hence, could not be included in the group. The condition in these latter families has been designated provisionally, as late onset familial CRC. The hereditary non-polyposis CRC families include 194 patients, 110 men and 84 women (mean age at diagnosis: 44 years; range: 16-74 years). Ninety two per cent of the patients were diagnosed by age 60. Colorectal cancer was diagnosed at progressively earlier ages in successive generations (p < 0.0005). The late onset CRC families comprised 30 patients, 20 men and 10 women (mean age at diagnosis: 62 years; range: 51-77 years). Fifty eight per cent of the CRCs in the patients belonging to the hereditary non-polyposis CRC families were located in the right colon compared with 13% in the late onset familial CRC group (p < 0.001). Multiple CRCs were found in 23% of the cases in the former but in only 3% in the latter families (p < 0.05). Adenomas associated with CRC were reported in 14.5% of the cases in the hereditary non-polyposis CRC families and in 30% of the cases in the late onset familial CRC group (p = NS). Extracolonic cancers, frequently encountered in hereditary non-polyposis CRC, were not found in the late onset CRC families. These findings indicate that there is a distinct clinical entity of non-polyposis CRC in which colorectal cancer develops at a more advanced age than in hereditary non-polyposis CRC. Future molecular genetic studies should confirm this hypothesis. In the meantime, recognition of these late onset familial CRC families, which will rest on clinical observations, is important because of the implications for the screening protocol.     

Reduction in length of stay for patients undergoing oesophageal and gastric resections with implementation of enhanced recovery packages

Introduction

The high mortality and morbidity associated with resection for oesophagogastric malignancy has resulted in a conservative approach to the postoperative management of this patient group. In August 2009 we introduced an enhanced recovery after surgery (ERAS) pathway tailored to patients undergoing resection for oesophagogastric malignancy. We aimed to assess the impact of this change in practice on standard clinical outcomes.

Methods

Two cohorts were studied of patients undergoing resection for oesophagogastric malignancy before (August 2008 – July 2009) and after (August 2009 – July 2010) the implementation of the ERAS pathway. Data were collected on demographics, interventions, length of stay, morbidity and in-hospital mortality.

Results

There were 53 and 55 oesophagogastric resections undertaken respectively for malignant disease in each of the study periods. The median length of stay for both gastric and oesophageal resection decreased from 15 to 11 days (Mann– Whitney U, p<0.001) following implementation of the ERAS pathway. There was no significant increase in morbidity (gastric resection 23.1% vs 5.3% and oesophageal resection 25.9% vs 16.7%) or mortality (gastric resection no deaths and oesophageal resection 1.8% vs 3.6%) associated with the changes. There was a significant decrease in the number of oral contrast studies used following oesophageal resection, with a reduction from 21 (77.8%) in 2008–2009 to 6 (16.7%) in 2009–2010 (chi-squared test, p<0.0001).

Conclusions

The introduction of an enhanced recovery programme following oesophagogastric surgery resulted in a significant decrease in length of median patient stay in hospital without a significant increase in associated morbidity and mortality.     

Serum levels of interferon-gamma, tumour necrosis factor-alpha, soluble interleukin-6R and soluble cell activation markers for monitoring response to treatment of leprosy reactions

Identifying pathogen and host-related laboratory parameters are essential for the early diagnosis of leprosy reactions. The present study aimed to clarify the validity of measuring the profiles of serum cytokines [interleukin (IL)-4, IL-6, IL-10, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha], the soluble IL-6 receptor (sIL-6R), soluble T cell (sCD27) and macrophage (neopterin) activation markers and Mycobacterium leprae-specific anti-PGL-I IgM antibodies in relation to the leprosy spectrum and reactions. Serum samples from 131 Indonesian leprosy patients (82 non-reactional leprosy patients and 49 reactional) and 112 healthy controls (HC) from the same endemic region were investigated. Forty-four (89.8%) of the reactional patients had erythema nodosum leprosum (ENL) while only five (10.2%) had reversal reaction (RR). Follow-up serum samples after corticosteroid treatment were also obtained from 17 of the patients with ENL and one with RR. A wide variability in cytokine levels was observed in the patient groups. However, IFN-gamma and sIL-6R were elevated significantly in ENL compared to non-ENL patients. Levels of IFN-gamma, TNF-alpha and sIL-6R declined significantly upon corticosteroid treatment of ENL. Thus, although the present study suggests limited applicability of serial measurement of IFN-gamma, TNF-alpha and sIL-6R in monitoring treatment efficacy of ENL, reactions it recommends a search for a wider panel of more disease-specific markers in future studies.