Comparison of total antibody and interferon-γ T-cell responses in patients following infection with brucellosis in Georgia.

Brucellosis is an ancient disease that still remains a significant threat to humans and is typically linked to exposure to infected animals and/or consumption of unpasteurized animal products. Despite this history, we have a relatively limited understanding of the host characteristics of this disease; consequently, further research is necessary. In this study, we examined the humoral immune response in 43 Georgian individuals that had been diagnosed with brucellosis 3-12 months before enrollment in the study, many of whom still had symptoms after the completion of antibiotic therapy. In total, 35 of 43 (83%) of the patients had antibodies that bound to Brucella lipopolysaccharide (LPS) by COMPELISA, and 34 of 38 (89%) patients had demonstrable specific antibodies to Brucellergene™ antigens; the results from the two ELISAs were highly correlated (p=0.031, r=0.851). We also studied the cellular immune responses in 15 patients. All of the patients generated interferon (IFN)-γ in response to ex vivo stimulation with Brucella protein antigens, and the majority of the patients maintained measurable humoral responses to both LPS and protein antigens. From this initial study, we conclude that measurement of antibody and of cellular (IFN-γ) responses to brucellergene OCB protein epitopes may be worthy of further investigation as an alternative or adjunct to current diagnostics.

Water-borne outbreak of oropharyngeal and glandular tularemia in Georgia: investigation and follow-up.

BACKGROUND: In November 2006, an outbreak of waterborne tularemia occurred in an eastern region in the Republic of Georgia. Outbreak investigation revealed 26 cases: 21 oropharyngeal and 5 glandular tularemia cases. METHODS: The presentation of the index case triggered an outbreak investigation involving the collection of clinical/ epidemiological data, application of tularine skin test, and laboratory confirmation of the possible cases using the tube agglutination test and polymerase chain reaction (PCR) testing. Serology results were verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. A case- control study along with follow-up was conducted 4 months after the index case presentation. RESULTS: Exudative pharyngitis, predominantly laterocervical adenitis, fever, and headache were the most prevalent clinical signs/symptoms observed. Depressed mood, concentration difficulties, and sleep disturbance were also detected. Bubo aspirates tested by PCR were positive in 4/4 cases and pharyngeal swabs also tested by PCR were positive in 2/3 cases. Francisella tularensis was isolated from the water samples. Comparison of the cases and controls did not reveal any statistically significant risk factors. A follow-up investigation revealed cases with protracted symptoms of fatigue, headache, and sleep disturbance. Additionally, 8/26 cases still had cervical adenopathy of prominent size. A delay in diagnosis was associated with persistent lymphadenopathy on follow-up examination (p = 0.05). CONCLUSION: We observed unique features of persistent neuropsychiatric symptoms and lymphadenopathy 5 months after tularemia infection which were associated with delayed diagnosis and the lack of prompt response to therapy. This outbreak of oropharyngeal tularemia emphasizes the importance of a rapid diagnostic and investigative response to tularemia. This type of response can prevent ongoing exposure, as well as provide expeditious treatment to mitigate persistent symptoms.

Maternal methadone use in pregnancy: factors associated with the development of neonatal abstinence syndrome and implications for healthcare resources.

OBJECTIVES: The objectives of this study were to investigate factors associated with the development of neonatal abstinence syndrome (NAS) and to assess the implications for healthcare resources of infants born to drug-misusing women. DESIGN: Retrospective cohort study from 1 January 2004 to 31 December 2006. SETTING: Inner-city maternity hospital providing dedicated multidisciplinary care to drug-misusing women. POPULATION: Four hundred and fifty singleton pregnancies of drug-misusing women prescribed substitute methadone in pregnancy. METHODS: Case note review. MAIN OUTCOME MEASURES: Development of NAS and duration of infant hospital stay. RESULTS: 45.5% of infants developed NAS requiring pharmacological treatment. The odds ratio of the infant developing NAS was independently related to prescribed maternal methadone dose rather than associated polydrug misuse. Breastfeeding was associated with reduced odds of requiring treatment for NAS (OR 0.55, 95% CI 0.34-0.88). Preterm birth did not influence the odds of the infant receiving treatment for NAS. 48.4% infants were admitted to the neonatal unit (NNU) 40% of these primarily for treatment of NAS. The median total hospital stay for all infants was 10 days (interquartile range 7-17 days). Infants born to methadone-prescribed drug-misusing mothers represented 2.9% of hospital births, but used 18.2% of NNU cot days. CONCLUSIONS: Higher maternal methadone dose is associated with a higher incidence of NAS. Pregnant drug-misusing women should be encouraged and supported to breastfeed. Their infants are extremely vulnerable and draw heavily on healthcare resources.

Visual evoked potentials in infants exposed to methadone in utero.

We investigated the effects of maternal drug misuse on neonatal visual evoked potentials (VEPs). Flash VEPs were recorded within 4 days of birth from 21 term infants of mothers misusing drugs and prescribed substitute methadone and 20 controls. Waveforms were classified as typical, atypical, immature or non-detectable, and amplitude and latencies were measured. VEPs from drug-exposed infants were less likely to be of typical waveform and more likely to be immature or non-detectable (p<0.01) than those of control infants. They were also smaller in amplitude (median 10.8 vs 24.4 microV, p<0.001). VEPs of drug-exposed infants had matured after 1 week but remained of lower amplitude than VEPs of newborn controls (p<0.01) and were non-detectable in 15%. Flash VEPs differ between maternal drug-exposed and non-drug-exposed newborns. Future research should address the specific effects of maternal methadone and/or other illicit drug misuse on infant VEPs, and associations between neonatal VEPs and subsequent visual development.

Neutralizing antibody response to booster vaccination with the 17d yellow fever vaccine.

A retrospective review was conducted of yellow fever vaccination among laboratory workers receiving annual serologic assessment to determine the initial and long-term response after boosting. Patients were divided into three groups based on pre-vaccination serology: Group 1, 1:10; Group 2, 1:20-1:40 and Group 3, >1:40. The percent with > or = four-fold increase in titers after booster vaccination were: 78% (646/829, Group 1), 65% (79/121, Group 2) and 10% (8/79, Group 3) (p<0.0001). The median times to titer failure (<1:40) were 798 days (Group 1), 3340 days (Group 2) and 7709 days (Group 3) (p<0.0001). Pre-vaccination serology influenced the initial and long-term response to yellow fever booster vaccination.

Candida krusei renal cyst infection and measurement of amphotericin B levels in cystic fluid in a patient receiving AmBisome.

Candida krusei is an opportunistic pathogen commonly implicated in urinary tract infections in immunocompromised patients. We present the first case of C. krusei renal cyst infection, occurring in a post-liver and kidney transplant patient with autosomal dominant polycystic kidney disease. Her persistent candiduria and fevers were refractory to prolonged therapy with AmBisome (Fujisawa Pharmaceuticals Co. Ltd., Osaka, Japan). She eventually required bilateral nephrectomies of her native kidneys. Cystic fluid was aspirated from six hemorrhagic and six nonhemorrhagic cysts. Cystic fluid cultures yielded C. krusei. Fluid from the nonhemorrhagic cysts was also analyzed for amphotericin B levels, measured using a bioassay. Free amphotericin B levels in the cysts were lower than the minimal inhibitory concentration for amphotericin B for this organism. We provide the first description of amphotericin B levels in cystic fluid obtained during bilateral nephrectomies.

Functional disturbance of marrow stromal microenvironment in the myelodysplastic syndromes.

The potential contribution of abnormal marrow stromal function to ineffective haemopoiesis in the myelodysplastic syndromes is unclear. We have compared the ability of stromal layers from normal (n = 7) and myelodysplastic (n = 9) marrow to alter proliferation and survival of the granulocyte-macrophage colony-stimulating factor/interleukin-3-dependent cell line F-36P. Co-cultures for 72 h in the absence of exogenous cytokines were either in direct contact with stroma or separated by transwell inserts. On normal stromal layers, the ratio of adherent F-36P cells relative to stromal cells increased from a mean of 0.2 +/- 0.01 (s.d.) at 4 h of co-culture to 0.34 +/- 0.08 after 72 h (n = 7). Corresponding values on myelodysplastic stroma (0.2 +/- 0.02 at 4 h and 0.35 +/- 0.05 at 72 h; n = 9) indicated that the ability of myelodysplastic stromal layers to regulate short-term proliferation of F-36P cells may be similar to normal. Apoptosis of F-36P cells was quantified after co-culture with normal or myelodysplastic stroma: results from myelodysplastic co-cultures were standardized as a fraction of values from co-cultures with paired normal stroma (apoptotic ratio). Augmented apoptosis of F-36P cells was detected in 8/9 co-cultures with myelodysplastic stroma (mean = 15.7 +/- 9.7%, n = 9), compared with corresponding normal stroma (mean = 12.4 +/- 4.6%, n = 7, P < 0.05) with a mean apoptotic ratio of 1.4 +/- 0.5 (P < 0.05). There was no correlation between stroma-related apoptosis and FAB type, tumour necrosis factor-alpha concentrations in the culture supernatant or numbers of stromal macrophages, and no evidence of involvement of the Fas pathway. Increased apoptosis was detected in cells grown in transwell inserts over stroma (23.8 +/- 3%, n = 5) compared to adherent cells in cultures with normal stromal layers, but this survival difference was not observed in co-cultures with myelodysplastic stroma. These results suggest that abnormal stromal function in patients with myelodysplastic syndromes may contribute to increased apoptosis of haemopoietic cells within the marrow microenvironment. The effect appears to be dependent on close cellular contact, rather than the release of soluble factors, but the exact mechanism remains unclear.

The effects of estrogen replacement therapy on airway function in postmenopausal, asthmatic women.

BACKGROUND: Data from multiple clinical, epidemiologic, and in vitro studies are conflicting regarding the effect of estrogen replacement therapy (ERT) on airway function in postmenopausal women with asthma. OBJECTIVE: To determine the impact of withdrawal of estrogen administration in postmenopausal, asthmatic women. METHODS: Twenty asthmatic women who were postmenopausal for at least 2 years and undergoing ERT were recruited for this prospective crossover study. Subjects continued taking baseline estrogen for 28 days, stopped taking estrogen for 28 days, and then resumed taking the medication for 14 days. Objective measurements were obtained by recording daily peak flows in the morning and evening and formal spirometry at days 14, 28, 42, 56, and 70. Compliance was measured by evaluating serum estradiol levels at days 28 and 56. Daily use of short-acting beta-agonist bronchodilators was also recorded. RESULTS: Differences in estradiol levels indicated compliance with the medication regimen. The combined day 14 and 28 (taking estrogen) mean percent predicted forced expiratory volume in 1 second (FEV(1)) was 77% compared with the combined day 42 and 56 (not taking estrogen) mean FEV(1) of 78% and the day 70 (taking estrogen again) FEV(1) of 76% (P>.05). Average peak flow measurements were 295.5 L/min for the duration of ERT, 293.9 L/min while not undergoing ERT, and 291.8 L/min when ERT was restarted for the final 2 weeks of the study (P>.05). Use of short-acting beta-agonist bronchodilators did not differ between study periods. CONCLUSION: These data indicate that neither the discontinuation nor reinitiation of ERT in postmenopausal, asthmatic women has any effect on objective measures of airway obstruction.

Assessment of stromal function, and its potential contribution to deregulation of hematopoiesis in the myelodysplastic syndromes.

BACKGROUND AND OBJECTIVES: The regulation of hematopoiesis by marrow stroma in vitro, has been shown to be abnormal in some patients with myelodysplastic syndromes (MDS). This study was performed to assess whether a range of mechanisms may be altered within the MDS microenvironment. DESIGN AND METHODS: The effects of diffusible factors produced by normal or MDS stromal layers on hematopoietic cells were studied by comparing the ability of media conditioned (CM) by normal or MDS stroma to regulate migration of target normal marrow CD34+ cells across 5 microm transmembranes. The ability of CM to stimulate hematopoietic cells was also assessed: changes in membrane polarity of KG-1a cells on exposure to stroma CM were compared. Subsequently, contact-mediated interactions between normal marrow CD34+ cells and normal and MDS stroma were studied: survival of allogeneic normal marrow CD34+ cells on live and glutaraldehyde-fixed normal and myelodysplastic stroma after 24h of co-culture was measured using 7-aminoactinomycin D staining. To determine whether hematopoietic cell survival on normal and MDS stroma was related to oxidative stress within the stromal microenvironment, intracellular superoxide levels, both constitutively and induced by tumor necrosis factor-a were measured within live stromal cells by FACScan analysis of ethidium bromide stained cells. RESULTS: The ability of CM from normal and MDS stroma to regulate short-term migration and activation of hematopoietic cells was similar. The mean percentage of apoptotic CD34+ cells (13+/-11%) adherent to glutaraldehyde-fixed myelodysplastic stroma was higher than on paired fixed normal stroma (11+/-10%) (n=6, p=0.056). Constitutive mean levels of superoxide in myelodysplastic cultures (9.5+/-2.1) were greater than in normal stromal cultures (4.9+/-0.6; n=6). However, following treatment with tumor necrosis factor-a, the mean value for superoxide in myelodysplastic stromal cultures was unchanged (fractional change=0.99+/-0.56), compared with an increase in normal stroma (fractional change=1.6+/-0.1, p<0.05). No correlation was observed between superoxide levels, proportion of apoptotic CD34+ cells and percentage of CD14+ stromal cells [mean 8%, range 0-37% (myelodysplastic); mean 1.3%, range 0-5% (normal)]. INTERPRETATION AND CONCLUSIONS: Abnormalities of stromal function in myelodysplastic syndromes are likely to be heterogeneous in origin: altered matrix molecules and changes in superoxide within stromal cells may contribute to abnormal survival and development of hematopoietic cells within the myelodysplastic marrow microenvironment

Timing of the appearance of multipotential and committed haemopoietic progenitors in peripheral blood after mobilization in patients with lymphoma.

The pattern of emergence of multipotential (CFU-A) and committed (CFU-GM and BFU-E) progenitor cells in peripheral blood has been examined in patients with Hodgkin's disease and non-Hodgkin's lymphoma. Mobilization protocols used chemotherapy with or without granulocyte colony-stimulating factor (n=8 and n=5, respectively). In all patients, the numbers of CFU-A, CFU-GM and BFU-E peaked simultaneously, rather than sequentially, suggesting that marrow regeneration after these mobilization protocols occurred from progenitors at all stages of differentiation. We conclude that peripheral blood stem cell harvest strategies based on peak values for total progenitor numbers will also capture maximum numbers of multipotential progenitors. However, the variable relationship between CFU-A and CFU-GM numbers suggests that overall progenitor cell numbers can give only a broad estimate of the absolute numbers of multipotential progenitors in an individual harvest.

Availability of smoking cessation resources for U.S. Army general medical officers.

Adequate resources are a necessity in providing effective smoking cessation interventions to active duty soldiers. The availability of smoking cessation resources was measured by a survey of Army general medical officers (GMOs). Questionnaires were successfully mailed to 232 Army GMOs, which was the entire population of this group in 1997. One hundred fifty GMOs (65%) returned the questionnaire voluntarily and anonymously. Fifty-three percent of GMOs identified the nicotine patch as a formulary item. All responders indicated that some form of nicotine replacement was available on formulary or for purchase in the area. Eighty-two percent of GMOs reported that group smoking cessation programs were available. The widespread availability of group smoking cessation programs may reflect an emphasis on this strategy by the Army's health promotion program. Additional attention needs to address the availability of nicotine replacement items and other adjunctive medications without cost to the beneficiary at military installations.

Efficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing.

BACKGROUND & AIMS: The optimal strategy for the detection of hereditary nonpolyposis colorectal cancer (HNPCC) gene carriers remains uncertain. We evaluated whether microsatellite instability (MSI) analysis or MSH2 and MLH1 protein immunostaining of tumors will screen individuals efficiently for germline MSH2 and MLH1 testing. METHODS: We performed a case-series study of 114 eligible families enrolled in our high-risk colorectal cancer (CRC) registry. Medical history data were collected on probands and relatives. MSI analysis was performed on proband tumors, and MSH2 and MLH1 protein immunostaining was assessed. Denaturing gradient gel electrophoresis was used to identify germline MSH2 or MLH1 mutations in probands found to have tumors with high-frequency MSI. RESULTS: Tumor tissue and adequate clinical data were available in 109 of the 114 families. Amsterdam criteria and Bethesda guidelines were met by 23% and 70% of the families, respectively. High-frequency MSI was identified in the proband tumors in 47 of the 109 families (43%). Germline MSH2 and MLH1 gene testing was carried out in the probands of 32 of 47 families with MSI-H tumors. Mutations were detected in 16 families (9 in MSH2 and 7 in MLH1) and sequence variants of uncertain significance in 5 families (1 in MSH2 and 4 in MLH1). Germline mutations or sequence variants of uncertain significance were detected in 15 of 19 (79%) of our Amsterdam families and in 6 of 13 (46%) of our non-Amsterdam families with MSI-H tumors. MSH2 and MLH1 protein immunostaining was assessed in 38 of the 47 MSI-H tumors. Unequivocal loss of hMLH1 expression was found in 20 tumors and loss of MSH2 expression in 9 tumors. Corresponding loss of protein expression was seen in 17 of 18 (94%) of tumors from probands with germline mutations or variants. CONCLUSIONS: The detection of high-frequency MSI or the loss of MSH2 or MLH1 immunostaining in CRCs are both useful criteria for selecting high-risk patients who should be tested for germline mutations in MSH2 or MLH1.

Subacute thyroiditis manifesting as fever of unknown origin.

Subacute thyroiditis (SAT) usually occurs in women in middle age with a viral prodrome, thyroid or neck tenderness, classic symptoms of thyrotoxicosis, and elevated erythrocyte sedimentation rate (ESR). We report a case in an 81-year-old man who initially had 2 days of fever to 101.2 degrees F, confusion, and bilateral lower extremity weakness. Extensive evaluation was remarkable only for the following laboratory values: thyrotropin (TSH) 0.02 microIU/mL, free thyroxine (FT4) 3.1 ng/dL, free triiodothyronine (FT3) 6.0 pg/mL, and ESR 98 mm/hr. One week later, the patient had persistent fevers to 102 degrees F; no source was found. The fever resolved, and 3 months later the patient had profound hypothyroidism (TSH >44.0 microIU/mL, FT4 0.4 ng/dL, ESR 13 mm/hr). A painless thyroid gland and atypical manifestations of hyperthyroidism are unusual in SAT. When fever is of unknown origin, SAT should be considered even if classic features are absent.

Glutathione S-transferase enzyme expression in hematopoietic cell lines implies a differential protective role for T1 and A1 isoenzymes in erythroid and for M1 in lymphoid lineages.

BACKGROUND AND OBJECTIVES: Glutathione S-transferases (GSTs) are phase II metabolizing enzymes which catalyze the conjugation of glutathione (GSH) to electrophilic substrates and possess selenium-independent glutathione peroxidase activity. The GST enzyme family includes the cytosolic isoforms GST-alpha, mu (GSTM), pi (GSTP), theta (GSTT) and sigma (GSTS). GSTT1, P1 and M1 are polymorphic and altered polymorphic frequency of genes encoding these proteins has been suggested as a potential risk factor for the development of hematopoietic malignancies. Overexpression of GSTs has also been implicated in chemotherapeutic drug resistance. This study was undertaken to elucidate the potential functional relevance of these genetic polymorphisms in hematopoiesis. DESIGN AND METHODS: GST genotype of 14 hematopoietic cell lines was determined by polymerase- chain-reaction (PCR). Gene expression of GSTs in a cell line was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on TaqMan 7700 and by semi-quantitative RT-PCR. Cytosolic GST protein expression was detected by Western blot. GST conjugation activity was assayed using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. RESULTS: GSTP1 expression was higher than other GSTs in 13/14 cell lines and paralleled CDNB conjugation activity. GSTP1 and GSTM1 predominated in lymphoid lines whilst T1 expression was relatively greatest in erythroid lines but was absent in 7/12 non-null lines. GSTT2 was expressed in only 3/4 lines. The 3 cell lines which expressed GSTA1 were all erythroid. INTERPRETATION AND CONCLUSIONS: Glutathione S-transerases showed differential lineage expression in hematopoietic cell lines. This implies a greater cytoprotective role for GSTT1 and GSTA1 in erythroid cells and GSTM1 in lymphoid cells. We postulate that inherited gene deletion of GSTT1 and M1 may produce increased genotoxic susceptibility for erythroid and lymphoid cell respectively, following exposure to xenobiotics that are substrates for these enzymes.

A survey of smoking cessation knowledge, training, and practice among U.S. Army general medical officers.

BACKGROUND: Historically, cigarette-smoking rates have been higher among military personnel than among civilians, although recently these rates have decreased. METHODS: In March 1997, a questionnaire assessing (1) training received on smoking cessation, (2) objective knowledge of smoking-cessation techniques, (3) frequency of practice habits, and (4) personal tobacco use among physicians, was successfully mailed to 232 of the total population of 279 Army general medical officers (GMOs). RESULTS: One-hundred-fifty (65%) GMOs returned questionnaires. Of these, 3.3% reported personal cigarette smoking, and 7.3% regularly used smokeless tobacco. During internship, few (13%) GMOs received smoking-cessation training. Primary care programs provided training more frequently than did surgery internship programs. The mean score on the objective knowledge portion was 72%. GMOs had a variable practice pattern in their use of smoking-cessation techniques (percent answering "usually" or "always"): helping patients set quit dates (35%), offering to prescribe the nicotine patch (59%), referring patients to a behavior-modification program (86%). Physicians who received training during internship were significantly more likely (p < 0.01) to help their patients set a quit date. Training did not result in a statistically increased frequency of other practice habits. CONCLUSIONS: GMOs received minimal training on smoking cessation during internship. GMOs refer patients to smoking-cessation classes, reflecting the strategy of the Army Health Promotion program. Strategies to increase the frequency that GMOs prescribe nicotine replacement and assist patients in setting a quit date are needed. Military smoking-cessation efforts may provide valuable lessons for the civilian community.

The role of the Sysmex SE9000 immature myeloid index and Sysmex R2000 reticulocyte parameters in optimizing the timing of peripheral blood stem cell harvesting in patients with lymphoma and myeloma.

Peripheral blood CD34/45+ cell (CD34/45) enumeration is an expensive and labour-intensive investigation but remains the standard assay for optimizing yield and timing of peripheral blood stem cell harvesting (PBSCH). The present study examined the value of the Sysmex SE9000 parameters (WBC, neutrophil count, and immature myeloid index (IMI)) and Sysmex R2000 reticulocyte parameters (absolute, high and medium fluorescence reticulocytes) in predicting the optimum timing of PBSCH in comparison to peripheral blood CD34/45. Sixty-four PBSCH from 23 patients with haematological malignancies were assessed with a variety of mobilization regimes used. Reticulocyte parameters showed high interpatient variability and did not prove clinically useful. IMI did not consistently predict satisfactory PBSCH yield except when > 1000 x 10(6)/l. Peripheral blood CD34/45 was the most useful predictor of yield. IMI > 20 x 10(6)/l was, however, a useful surrogate for predicting a rise in peripheral blood CD34/45 from nadir and proved to be superior to WBC or neutrophil count. A rising IMI is a marker of early regeneration and has a role in determining when to initiate enumeration of peripheral blood CD34/45.

Poor response rate to a continuous schedule of Amifostine therapy for 'low/intermediate risk' myelodysplastic patients.

Ineffective haemopoiesis leading to cytopenia presents the major clinical management problem for patients with myelodysplasia (MDS). Preliminary studies have demonstrated that the synthetic aminothiol Amifostine stimulates multilineage haemopoiesis both in vitro and in vivo in patients with MDS. We have treated 12 patients with an uninterrupted 8-week schedule of thrice-weekly intravenous Amifostine with a starting dose of 300 mg/m2 escalating to 450 mg/m2 in non-responders. No patients satisfied response criteria on study but two patients showed minor responses. We conclude that therapeutic response to Amifostine in MDS may be schedule dependent.