Changes in cellular structure of heat-treated Salmonella in low-moisture environments.

AIMS: Salmonella cells desiccated in an environment with low-water activity (aw ) show longer survival times and enhanced resistance to heat. However, little is known about the cellular ultrastructure of Salmonella in low-aw environment in relation to the survival and persistence during desiccation. MATERIALS AND RESULTS: In this study, Salmonella Enteritidis strain PT30 was dehydrated by exposure to air or by mixing with wheat flour (aw 0·30 at room temperature) for 7 days followed by heat treatment at 80°C for 10, 20, 60 min respectively. Transmission electron microscopy (TEM) was employed to examine and compare the ultrastructure of heat-treated S. Enteritidis cells after desiccation with the cells suspended in trypticase soy broth (TSB). Cells suspended in TSB broth showed disrupted ribosomes, congregated proteins and denatured DNA. However, no significant alterations were observed in the ultrastructure of the desiccated cells after heat treatment. The number of desiccated S. Enteritidis cells decreased by <1·5 log CFU per gram after 80°C treatment for 60 min, however, cells suspended in TSB declined more than 5 log10 CFU per mL at 80°C within 5 min. CONCLUSIONS: A drastic difference in the number of survivors and cellular ultrastructure was observed between vegetative and air or food-dried S. Enteritidis cells after subjecting to heat treatment at 80°C. No significant ultrastructure changes were observed in desiccated cells after heat treatment except for roughening and corrugating surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a direct comparison to illustrate how desiccation influences the cell ultrastructure before/after heat treatment, which will aid in better understanding of the fundamental mechanism underlying the increased thermal resistance of Salmonella cells in low-aw environment.

Rapid differentiation of Mycobacterium bovis and Mycobacterium tuberculosis based on a 12.7-kb fragment by a single tube multiplex-PCR.

The aim of this work was the design and validation of a rapid and easy single tube multiplex-PCR (m-PCR) assay for the unequivocal differential detection of Mycobacterium bovis and Mycobacterium tuberculosis. Oligonucleotide primers were based on the uninterrupted 229-bp sequence in the M. bovis genome and a unique 12.7-kb insertion sequence from the M. tuberculosis genome, which is responsible for species-specific genomic polymorphism between these two closely related pathogens. The m-PCR assay was optimized and validated using 22 M. bovis and 36 M. tuberculosis clinical strains isolated from diverse host species and 9 other non-tuberculous mycobacterial (NTM) strains. The designed primers invariably amplified a unique 168-bp (M. bovis-specific) and 337-bp (M. tuberculosis-specific) amplicon from M. bovis and M. tuberculosis strains, respectively. The accuracy of the assay, in terms of specificity, was 100%, as none of the NTM strains tested revealed any amplification product. As little as 20 pg of genomic DNA could be detected, justifying the sensitivity of the method. The m-PCR assay is an extremely useful, simple, reliable and rapid method for routine differential identification of cultures of M. bovis and M. tuberculosis. This m-PCR may be a valuable diagnostic tool in areas of endemicity, where bovine and human tuberculosis coexist, and the distinction of M. bovis from M. tuberculosis is required for monitoring the spread of M. bovis to humans.

A multiplex-PCR for the differentiation of Mycobacterium bovis and Mycobacterium tuberculosis.

A multiplex-polymerase chain reaction (PCR) assay based on one-step amplification and detection of two different mycobacterial genomic fragments was designed for differentiation of Mycobacterium bovis and Mycobacterium tuberculosis. The oligonucleotide primers were chosen from a 500-bp genomic fragment which is well conserved in M. bovis and the pncA gene (based on M. tuberculosis-specific nucleotide polymorphism, a cytosine residue at position 169), specific for M. tuberculosis. The multiplex-PCR allowed detection of a single product of 500 bp in M. bovis isolates while M. tuberculosis isolates generated a single product of 185 bp, with or without an additional product of 500 bp. None of the atypical mycobacterial isolates revealed any amplification products. The method was found to be highly specific and could detect as little as 20 pg of pure DNA. This multiplex-PCR assay, based on the 500-bp fragment and the pncA gene, may be very useful for the rapid and specific differentiation of these two closely related mycobacteria and easy to use in medical and veterinary microbiological laboratories.

Discovery of potent and selective phenylalanine derived CCR3 receptor antagonists. Part 2.

Highly potent CCR3 antagonists have been developed from a previously reported series of phenylalanine ester-based leads. Solution-phase, parallel synthesis optimization was utilized to identify highly potent, functional CCR3 antagonists.

Metal mediated protease inhibition: design and synthesis of inhibitors of the human cytomegalovirus (hCMV) protease.

A versatile synthetic route to a novel series of bis-imidazolemethanes designed to inhibit the hCMV protease has been developed and a series of potential metal binding inhibitors has been identified. In selectivity assays, the compounds were highly specific for CMV protease and showed no inhibition (IC50 > 100 microM) of other prototypical serine proteases such as trypsin, elastase, and chymotrypsin. Although the presence of free zinc ions was found to be an absolute requirement for the in vitro biological activity of this class of inhibitor, the potency of the inhibitors could not be improved beyond the micromolar level.

Pulmonary metastases in children and adolescents with well-differentiated thyroid cancer.

UNLABELLED: In this study, 27 patients less than 18 yr old with pulmonary metastases from well-differentiated thyroid carcinoma were evaluated to determine their response to (131)I therapy. METHODS: Of 121 children and adolescents treated with (131)I between 1963 and 1996, 27 patients had pulmonary metastases associated with nodal disease. Treatment response from (131)I was measured by three parameters: chest radiograph, scintigraphic images and serum thyroglobulin levels. Total activity of (131)I administered ranged from 4.6 GBq (125 mCi) to 38.7 GBq (1.05 Ci). Four patients were given one treatment, 8 were given two treatments, 4 were given three treatments and 11 were given more than three treatments. Radiation doses to the lungs were estimated in 14 patients using the MIRD methodology. The minimum duration of follow-up was 6 mo. RESULTS: At the time of initial presentation, diagnostic (131)I studies revealed bilateral radioiodine uptake in the lungs in 19 (70.4%) patients, whereas 12 (44.4%) patients had abnormal chest radiographs. One patient was lost to follow-up and was excluded from the study. Of the 26 patients studied, complete ablation of pulmonary metastases was observed in 8 (30.8%), partial ablation in 17 (65.4%) and there was no response to treatment in 1 (3.8%). Dosimetric parameters such as radioiodine uptake as a percentage of therapeutic activity, effective half-life and radiation dose delivered to the lungs were evaluated with each therapy. There was a progressive decline in each of these parameters with successive therapies. No correlation was observed between the radiation dose delivered and the response of pulmonary metastases to therapy. The number of therapies and amount of radioiodine administered had no influence on the ablation response. Of the 26 patients, 13 had a follow-up duration of less than 5 yr, 7 had 5-10 yr and 6 had more than 10 yr. One patient developed new metastases after 7 yr of diagnosis and treatment. One patient died of the disease after 4 yr. All surviving patients were asymptomatic and leading normal lives. CONCLUSION: Complete response of pulmonary metastases after (131)I therapy is difficult to achieve. A partial response with reduction of metastatic disease is possible and, in general, the patients had a good quality of life with no further disease progression and a low mortality rate.

Effect of chronic treatment with amlodipine in non-insulin-dependent diabetic rats.

We have investigated the effects of amlodipine on streptozotocin-(STZ) induced neonatal non-insulin-dependent diabetes mellitus (NIDDM) rats. NIDDM was induced by intraperitoneal injection of STZ (70 mg kg-1) to 5-day-old rat pups. The animals were weaned at 30 days and maintained with food and water ad libitum for 3 months. Amlodipine (5 mg kg-1 p.o.) was administered for 6 weeks after the animals were confirmed diabetic (3 months after the STZ injection). A group of control animals were also maintained and this group received citrate buffer 5 days after birth. Fasting- and fed-glucose levels in NIDDM rats were significantly higher than control rats. Treatment with amlodipine reduced the elevated fasting- and fed-glucose levels significantly. Results of the oral glucose tolerance test (OGTT) revealed the glucose tolerance is impaired in the NIDDM rats. There was a marked increase in glucose levels after oral administration of glucose in the control NIDDM rats. Increased glucose levels were found to be associated with increased insulin levels. Treatment with amlodipine in the NIDDM rats caused a decrease in insulin release, however, glucose levels were found to be lowered significantly indicating that amlodipine causes an increase in insulin sensitivity. In conclusion, our data indicated that amlodipine increases insulin sensitivity in neonatal-STZ NIDDM rats.

Benzothiopyran-4-one based reversible inhibitors of the human cytomegalovirus (HCMV) protease.

A novel class of CMV protease inhibitors based on a benzothiopyran-S,S-dioxide nucleus has been discovered. Enzyme kinetic data supports a reversible mode of inhibition for a representative member of this class, 2-(3-pyridyl-N-oxide)benzothiopyran-4-one-S,S-dioxide, 1. Experiments in the presence and absence of the disulfide reducing agent DTT suggest that the inhibition by 1 is not due to oxidative inactivation of the enzyme. Also presented are results of some SAR studies of the benzothiopyranone ring system.

Adoptive immunotherapy of murine lymphosarcoma: comparative study using allo-immune or tumor-immune T cells.

The antitumor effect of allosensitization with lymphocytes and skin graft of DBA/2 mice was evaluated using immunogeneic, transplantable Lymphosarcoma (LS-A) syngeneic to Swiss mice. A dose dependent tumor inhibitory effect in terms of tumor free mice was observed in mice sensitized i.p. with lymphocyte doses between 10-100 million per animal. Sensitization with allogeneic primary skin graft was more effective than lymphocyte immunization. The antitumor immunity could be adoptively transferred in syngeneic Swiss mice using either allo-immune or tumor-immune T cells. Analysis of T cell phenotypes using monoclonal antibodies against cell surface markers CD4 and CD8, indicated absolute dependence on the CD4+ T cells subset in tumor cure in case of allo-immune as well as tumor-immune T cells. CD8+ T cell subset was found essential only in case of allo-immune T cell therapy. Immunosuppression of mice with whole body gamma irradiation (4 Gy), 6 hr before transfer of allo-immune or tumor-immune T cells did not abrogate the therapeutic ability of allo-immune or tumor-immune T cells. Our results suggest that allosensitization could be an effective method of generating effector lymphocyte populations that might be used to treat tumors that exhibit detectable immunogenecity.

Brain metastases in well-differentiated carcinoma of the thyroid.

Fifteen patients (4 males and 11 females) developed brain metastases from well-differentiated thyroid cancer within 1 month to 14 years of the initial diagnosis. One patient presented with a brain tumor. Except for 3 patients with unique brain metastases, all the others had extensive metastases in nodes, lungs and bones in various combinations. Brain metastases generally appeared after the onset of metastases at other sites. The histology of the brain tumor matched the primary pathology in the 6 operated cases. The treatment was surgery and external radiation in 6 cases, and radioiodine or chemotherapy in the others. Survival in general was less than 6 months after the diagnosis of brain metastases. The prognosis is poor once the onset of brain metastases is evident.

Metastasis to the liver in well-differentiated carcinoma of the thyroid.

Eleven patients (6 women; 5 men) of a series of 1,916 developed liver metastasis from differentiated thyroid cancer within 3 months to 202 months after the initial diagnosis. Liver metastasis generally appeared after the onset of metastases at other sites. The metastasis to the liver was clinically suspected in 8 patients whereas it was accidentally diagnosed on ultrasound examination of abdomen for a complaint of back pain in 1 patient, on a whole body diagnostic radioiodine scan in 1 case, and on radionuclide liver scan in 1 patient. Three patients had noniodide concentrating hepatic metastasis which were treated with chemotherapy but with poor response. The remaining 8 patients were treated with radioiodine. The survival rate was poor but could not be attributed to liver metastasis per se because of the extensive metastatic disease at other sites.

Epidemiological studies in children of a low-endemic region, a high-endemic region, and dwellers of a leprosy colony: evaluation of anti-ND-BSA antibodies and lepromin response.

Children residing in a low-endemic region (LER), a high-endemic region (HER), and a leprosy colony contact population (CP) were evaluated for lepromin response as well as reactivity to the Mycobacterium leprae-specific synthetic antigen, ND-BSA. The mean reactivity to ND-BSA in the LER group (OD 0.03 +/- 0.03, N = 71) was significantly lower (p < 0.001) than that in the contact population (OD 0.14 +/- 0.09, N = 140) as well as the population residing in the HER (OD 0.09 +/- 0.08, N = 1340). ELISA-positive results were the highest (21.4%) with the CP group and lowest (0.0%) in the LER group, suggesting that it was a measure of the extent of exposure of M. leprae. In the contact population, females showed a preponderance for ELISA positivity over males (p < 0.005), a finding not observed with the HER population. The Mitsuda responses showed a Gaussian-type distribution in all of the three populations examined with the mean response being highest in the LER (6.0 mm +/- 2.9) and lowest in the HER (4.5 mm +/- 2.0) groups. The percent positivity for the Mitsuda reaction was found to be highest in the LER (93.0%) and lowest in the HER (88.3%) groups. The Mitsuda response thus appears to be independent of M. leprae exposure, and its interpretation in a given population needs consideration of several factors, such as nutritional, environmental, etc.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical utility of serum thyroglobulin in metastatic disease.

We examined the usefulness of serum thyroglobulin (Tg) levels in 196 patients with metastatic disease. Of these, 51 patients had a thyroidal primary (40 differentiated, 7 medullary and 4 undifferentiated), 35 patients had a nonthyroidal primary and in 110 patients the primary site was not known. Serum Tg was raised in 74.5 per cent (38 of 51) patients with carcinoma of the thyroid and in 92.5 per cent (37 of 40) patients with differentiated thyroid carcinoma (DTC). Twelve (34.3%) patients with proved nonthyroidal malignancies and 34 (30.9%) patients with unknown primary origin had elevated serum Tg levels. Of the 110 patients with unknown primary site thyroidal primary in 10 patients (all with elevated serum Tg levels) and nonthyroidal primary in 38 patients (7 had raised serum Tg levels) could be established. The sensitivity (for DTC) and the specificity (for nonthyroidal primary) of serum Tg estimation were 94.0 per cent (47 of 50) and 74.0 per cent (54 of 73) respectively and for patients with distant metastases were 100 (29 of 29) and 85.1 per cent (40 of 47) respectively. The positive and the negative predictive values of serum Tg were 71.2 (47 of 66) and 94.7 per cent (54 of 57) respectively and for patients with distant metastases were 80.6 (29 of 36) and 100 per cent (40 of 40) respectively. Our findings suggest that for patients presenting with metastases (particularly distant metastases) of an unknown primary site, serum Tg estimation is of great value to identify or rule out the involvement of the thyroid as the primary organ.

Conformationally constrained peptides and semipeptides derived from RGD as potent inhibitors of the platelet fibrinogen receptor and platelet aggregation.

Structure-activity studies have been pursued on cyclo-S,S-[Ac-Cys-(N alpha-Me)Arg-Gly-Asp-Pen]-NH2, 2 (SK&F 106760), a potent inhibitor of platelet aggregation, in an effort to improve potency and affinity for the GPIIb/IIIa receptor. Modifications on the N- and C-termini of 2 produced a series of peptides which indicate that the C-terminal carboxylate group may be a secondary receptor-binding element. Further modification by replacing the disulfide tether N alpha-acetylcysteine/penicillamineamide with the novel, inexpensive, achiral, constrained, and more lipophilic tether 2-mercaptobenzoyl/2-mercaptoaniline (Mba/Man) afforded the semipeptide cyclo-S,S-[Mba-(N alpha-Me)Arg-Gly-Asp-Man], 18 (SK&F 107260), which exhibited significant enhancement in both affinity and potency. To further investigate the effect of the phenyl ring at the C-terminus, peptides bearing the novel (2R,3S)- and (2R,3R)-beta-phenylcysteines were synthesized, which culminated in the cyclo-S,S-[Ac-Cys-(N alpha-Me)Arg-Gly-Asp-(2R,3S)-beta-phenylCys]-OH peptide, 22, which displayed substantial affinity and potency. We describe, herein, the development of both 18 and 22 and the additional structural modifications within the constrained cyclic disulfide ring to probe the stereochemical and steric requirements for receptor interaction.

Significance of antithyroglobulin autoantibodies in differentiated thyroid carcinoma.

The incidence of antithyroglobulin autoantibodies (ATA) was 17.7% in 963 patients (who attended the clinic from 1981 to 1990) with differentiated thyroid carcinoma (DTC). Another 12 patients developed ATA for a transient period after the treatment with radioiodine. The prevalence of ATA in females (21.5%, 123/572) was significantly higher (p < 0.001) than that seen in males (12.0%, 47/391). Age-dependent occurrence of ATA was not seen for the various age decades. The ATA was more prevalent (p < 0.01) with the papillary type of tumor (118/564) as compared to the follicular variety (51/398). ATA did not influence the metastatic spread of the tumor at the initial presentation (105/170 for the ATA-positive group and 445/793 for the ATA-negative group). However, within the group with metastases, 82.9% (87/105) of patients had local spread into the neck in the presence of ATA, which was significantly higher (p < 0.01) than that seen for patients without ATA (63.8%, 284/445). For assessment of the influence of ATA on the outcome of the disease, the data from 222 patients (46 positive and 176 negative for ATA), with a minimum follow-up of 5 years (mean follow-up of 7.4 years), was considered suitable for analysis. The outcome of the disease was comparable in the presence and the absence of ATA (38/46 and 137/176 patients became disease-free in ATA-positive and -negative groups, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

An immunoradiometric assay for measurement of serum thyroglobulin.

A two-site immunoradiometric assay (IRMA) for estimation of serum thyroglobulin (tg) is optimized using partially purified rabbit anti-tg antibodies (ammonium sulphate precipitated) as a solid phase antibody and affinity purified 125I labeled anti-tg antibodies as a tracer. The IRMA correlated well (r = 0.93, n = 200, P < 0.001) with conventional radioimmunoassay (RIA). The inter- and intra-assay coefficient of variation were comparable for both methods. The IRMA method gave a superior sensitivity and working range (2 ng/ml, 3-1000 ng/ml) as compared to that of RIA method (6 ng/ml, 9-500 ng/ml). Using IRMA the total incubation time was curtailed to 4 h as compared to 90 h required for conventional RIA. The shelf-life of tracer antibody used in IRMA was much longer (3 months) as compared to tracer tg (3-4 wk) used in RIA. The optimized IRMA for quantitation of serum tg has superior assay characteristics as compared to RIA.

A simple radioassay for detection of antithyroglobulin autoantibodies.

Radioassay technique for detection of anti-thyroglobulin autoantibodies (ATA) in serum samples using protein-A rich Staphylococcus aureus as an immune complex separating agent was comparable (r = 0.99; n = 42; P < 0.001) to that evaluated with anti-human gammaglobulin (AHGG). The inter- and intra-assay coefficient of variations were lower (7.4 and 2.2% respectively) using Staph. aureus as compared to that observed with AHGG (10.4 and 4.7% respectively). A highly significant correlation was observed (r = 0.53; n = 71; P < 0.001) between per cent bound radiolabel thyroglobulin in radioassay and log reciprocal titre of ATA by haemagglutination method. Radioassay scored more number of positive sera as compared to haemagglutination method for normal controls (6 vs 1; n = 46) and patients of thyroid diseases (151 vs 70; n = 238). Due to the relatively poor stability of tracer on storage larger inter-assay coefficient of variations were observed with the radiolabelled preparations older than 10-12 days. The radioassay method evaluated is sensitive, reproducible and useful in detecting presence of anti-thyroglobulin autoantibodies.

Monoclonal antibodies to human thyroglobulin: evaluation of immunoreactivity.

We have earlier reported production and characterization of monoclonal antibodies (MAbs) to human thyroglobulin (h-tg). In the present study H10 I MAb was evaluated for its immunoreactivity towards different forms of tg and various human thyroid tumours. The specificity of H10 I MAb was validated by the absence of cross reaction with tri-iodothyronine (T3) Thyroxine (T4) and human gamma globulins. Sodium-dodicyl-sulphate polyacrylamide gel electrophoresed (SDS-PAGE) immunoblot of h-tg on the nitrocellulose membrane revealed multiple immunoreactive bands on reaction with polyclonal antibody (PAb) in comparison with total lack of reactivity with H10 I MAb. The absence of immunoreactivity of H10 I MAb was demonstrated with SDS treated, Dithiothreitol (DT) treated and heat denatured tg using dot immunobinding technique. However, the H10 I MAb was able to react with tg treated with unfolding agents such as urea and guanidine hydrochloride. All the treated forms of tg were equally recognized by PAb. The immunoreactivity of the oxidized/reduced tg towards H10 I MAb was markedly reduced (60.0%) as compared to that obtained with native tg. It appears that H10 I MAb is directed towards conformational epitope involving sulphydryl bonds. Immunohistochemically, a comparable immunoreactivity between PAb and MAb was observed with normal thyroid tissues, follicular thyroid tissues, Hurthle cell carcinoma tissues and poorly differentiated thyroid tumor tissues using immunoperoxidase staining. The sections from papillary carcinoma tissue (thyroid as well as metastatic lymph node) exhibited intense immunoreactivity with PAb. Thyroglobulin present on these sections was not recognized by H10 I MAb. Nonetheless, H10 I MAb was able to detect tg in follicular differentiation wherever present. The absence of immunoreactivity of H10 I MAb in papillary carcinoma strongly suggests that this neoplasm produces tg which is antigenically different from the protein present in the normal tissue. The reactivity of H10 I MAb with metastatic lymph node of an unknown primary origin suggests its usefulness in the identification of prevalent metastasis of differentiated thyroid carcinoma other than papillary type.