TB disease patterns by HIV and diabetes status.

BACKGROUND: TB is commonly categorised as pulmonary (PTB) or extrapulmonary TB (EPTB). Knowledge of TB disease patterns (PTB and/or EPTB) and determining risk factors remains limited.METHODS: This was a prospective cohort study using point-of-care ultrasound (POCUS) in Indian patients with presumed TB. Clinical and imaging data were used to develop refined case definitions for PTB, concurrent PTB and EPTB (PTB + EPTB) and EPTB without PTB (EPTB). These groups were analysed by HIV (HIV+/-) and diabetes mellitus (DM+/-) status.RESULTS: Of 172 HIV-/DM- patients with TB, 48% had PTB, 23% PTB + EPTB and 29% had EPTB, totalling 52% with any EPTB (PTB + EPTB or EPTB). In HIV+/DM- patients with TB (n = 35), 6% had PTB, 40% had PTB + EPTB and 54% had EPTB, accounting for 94% with EPTB. In HIV-/DM+ patients with TB (n = 61), 61% had PTB, 28% had PTB + EPTB and 11% had EPTB, representing 39% with EPTB.CONCLUSION: Refined case definitions revealed high proportions of EPTB even without HIV or DM. HIV further altered the TB disease pattern towards EPTB and DM towards PTB. Therefore, the dichotomy between PTB or EPTB does not represent the actual spectrum of TB disease. EPTB should receive higher priority in research and clinical practice.

Effect of increased glucose levels on short-term outcome in hypertensive spontaneous intracerebral hemorrhage.

OBJECTIVE: Spontaneous intracerebral hemorrhage (ICH) can be a devastating event. Increased glucose levels in the plasma may be related to poor outcomes; however, the precise association remains unclear. METHODS: We retrospectively assessed 116 patients with hypertensive ICH. Glucose level in the plasma was assessed at days 0, 1, and 3. Outcome variables were mortality within 7 and 30days and the National Institutes of Health Stroke Scale (NIHSS) score at day 14 after ICH onset. RESULTS: Twenty deaths had occurred by day 7, and the 30-day mortality rate was 31.9%. Hyperglycemia at day 0 was significantly more common in patients who died within 7days or 30days. Hyperglycemia at day 1 was more common in patients with an NIHSS score >15 on admission and at day 14. No differences in glucose levels were found between diabetic and non-diabetic patients. Among non-diabetic patients, higher glucose levels were related to poorer outcomes (death or an NIHSS score >15). In multivariate analysis, glucose levels >140mg/dL at day 1 were related to the 30-day mortality (hazard ratio=2.65; 95% confidence interval [CI]=1.15-6.12, p=0.02), and glucose levels >160mg/dL at day 1 were associated with an NIHSS score >15 at day 14 (odds ratio=3.08; 95% CI=0.9-10.5, p=0.07). White blood cell counts were directly associated with poorer outcomes and significantly correlated to glucose levels. CONCLUSION: Initially increased glucose levels and increased levels within 24h of ICH onset were related to poorer outcomes. Altered glucose metabolism may be due to inflammatory cell activation. Further studies are needed to clarify the association between immune activation and glucose metabolism after ICH onset.

The 2009 pandemic influenza A(H1N1) coincides with changes in the epidemiology of other viral pathogens causing acute respiratory tract infections in children.

BACKGROUND: In Germany, the outbreak of the novel pandemic 2009 influenza A(H1N1) virus A(H1N1)pdm09 caused a wave of high activity between November 2009 and January 2011. The aim of this study was to investigate the prevalence of 19 respiratory pathogens in children hospitalized for lower respiratory tract infections during the winter influenza seasons of 2009/2010 and 2010/2011 and to observe a possible impact of influenza A(H1N1)pdm09 on the epidemiology of other epidemic viruses. MATERIALS AND METHODS: Specimens were nasopharyngeal aspirates which had been collected from children admitted to the participating hospitals in the area of Mainz, Wiesbaden, and Kiel, Germany, with acute community-acquired lower respiratory tract infections. The specimens were subjected to a previously described multiplex reverse transcription PCR assay to detect the following microorganisms: enterovirus, influenza virus types A and B, respiratory syncytial virus (RSV), parainfluenzavirus types 1-4, adenovirus, Mycoplasma pneumoniae, Chlamydophila pneumoniae, rhinovirus, human metapneumovirus (hMPV), coronavirus OC43 and 229E, influenza A(H1N1)pdm09, Bordetella pertussis, Bordetella parapertussis, and Legionella pneumophila. RESULTS: A total of 3,998 clinical specimens were collected from July 2009 to March 2011, of which 296 were positive for A(H1N1)pdm09. An epidemic of seasonal influenza A or B was not observed in the 2009/2010 season, but a minor epidemic of seasonal influenza B was observed in January/February 2011. Influenza A(H1N1)pdm09 coincided with the absence of the seasonal influenza A of former years. The RSV and hMPV epidemics of 2009/2010 erupted several weeks later than expected based on data collected in the PID-ARI-Network during the past 10 years, whereas in the 2010/2011 influenza season they occurred as expected. CONCLUSIONS: The emergence of the novel influenza A(H1N1)pdm09 virus may have been influenced the epidemiology of other epidemic viruses, such as the RSV and hMPV. No epidemic of seasonal influenza was observed in the 2009/2010 influenza season.

Continued statin therapy could improve the outcome after spontaneous intracerebral hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) often represents a devastating event despite maximal therapeutic efforts. Statins are drugs primarily used as cholesterol reducers with several pleiotropic effects that may result in neuroprotection. In this study, we assessed the continued use of statins after acute ICH. From January 2008 to October 2010, we analyzed a retrospective cohort of 178 patients with acute ICH. Patients with head injury, cerebral tumors, hemorrhage after ischemic stroke, and having a National Institute Health Stroke Scale (NIHSS) score of greater than 30 points on admission were excluded. In 29 patients, statins were continued within the first 24 h after onset of ICH and, subsequently, given daily until discharge, whereas 149 nonusers were used as controls. Inpatient mortality, NIHSS, and Glasgow Outcome Score (GOS) at discharge as well as mortality after 10 days, 3 months, and 6 months were recorded as outcomes. Additionally, changes of C-reactive protein (CRP) and white blood cell (WBC) counts, as well as aspartate transaminase and alanine transaminase levels were assessed. Except for the number of hypertensive and diabetic patients, characteristics on admission were similar between both groups. No mortality was observed in statin users, whereas 19 controls (12.7 %) died (p = 0.04) until discharge; after 10 days, 3 months, and 6 months, a similar trend was found. After 6 months, statin use was associated to lower mortality in regression models (OR = 0.32, 95 % CI = 0.11-0.95, p = 0.04). In the same way, statin use was related to NIHSS reduction (-3.53, 95 % CI = -7.59 to 0.42, p = 0.07). In mixed models, changes of WBC counts and CRP levels were associated with statin use. The hepatic enzymes were similar between groups. The continued use of statins after ICH could be associated to early neurological improvement and may reduce mortality within 6 months. Immunomodulation as a pleiotropic effect of statins may represent one of the underlying mechanisms.

Generation of cellular immune responses to HCV NS5 protein through in vivo activation of dendritic cells.

Chronic hepatitis C (HCV) infection is a substantial medical problem that leads to progressive liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The aim of this study was to achieve sustained cellular immune responses in vivo to a HCV nonstructural protein using dendritic cell (DC)-based immunization approach. We targeted the HCV NS5 protein to DCs in vivo by injecting microparticles loaded with this antigen. The DC population was expanded in BALB/C mice (H-2(d) ) by hydrodynamic injection of a plasmid pUMVC3-hFLex expressing the secreted portion of the human Fms-like tyrosine kinase receptor-3 ligand (hFlt3). Mice were subsequently injected with microparticles coated with HCV NS5 protein via the tail vein. Cellular immune responses were determined with respect to secretion of INFγ and IL2 by CD4(+) cells and cytotoxic T-lymphocyte (CTL) assays in vitro; inhibition of tumour cell growth was employed for the assessment of CD8(+) generated activity in vivo. We found that Flt3L treatment expanded the DC population in the spleen to 43%, and such cells displayed a striking upregulation of CD86 as well as CD80 and CD40 co-stimulating molecules. Viral antigen-specific T(H) 1 cytokine secretion by splenocytes was generated, and CTL activity against syngeneic NS5 expressing myeloma target cells was observed. In addition, these cells inhibited tumour growth indicating that NS5-specific robust CTL activity was operative in vivo. Thus, the capability of activating DCs in vivo using the methods described is valuable as a therapeutic vaccine strategy for chronic HCV infection.

Transmission of feline calicivirus via the cat flea (Ctenocephalides felis).

In this study, a possible role of the cat flea (Ctenocephalides felis) in transmitting feline calicivirus (FCV) was examined. Fleas were fed via artificial membranes with FCV-spiked bovine blood, free of anti-FCV antibodies. Flea feces were collected daily for 10 days and incubated at room temperature. Infectivity of the feces was tested in vitro using Crandell-Reese Feline Kidney (CRFK) cells. FCV remained infectious for 8 days. These flea feces were also used to oronasally inoculate four specific pathogen-free (SPF) kittens. All kittens were successfully infected as demonstrated by virus isolation from pharyngeal swabs and seroconversion. Two of the cats showed, in addition, clinical signs. Besides the infection of cats with flea feces containing FCV, four SPF kittens were exposed to fleas that were fed with FCV-spiked bovine blood. One of the kittens was successfully infected via this route as demonstrated by virus isolation from pharyngeal swabs and virus isolation. The results of this study show that fleas can spread infectious virus through their feces or by stitch and must be considered a source of infection for uninfected cats.

TSST-1 induces Th1 or Th2 differentiation in naïve CD4+ T cells in a dose- and APC-dependent manner.

Superantigens are potent activators of the immune system, causing a variety of diseases, ranging from food poisoning to septic shock. Here, we examined the effects of different toxic shock syndrome toxin 1 (TSST-1) concentrations on the activation, proliferation and synthesis of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in purified naïve human CD4+ T cells in a serum-free in vitro system. TSST-1 given in low doses (1-10 pg/ml) generates a pronounced T helper 2 (Th2)-like cytokine profile, characterized by elevated IL-4-expressing T-cell populations and reduced IFN-gamma-producing populations, whereas higher doses (100 pg/ml) induce a Th1-like profile, with increased expression of IFN-gamma and reduced expression of IL-4. These patterns were even more pronounced by adding exogenous cytokines like IL-12 and IL-4 and by the type of antigen-presenting cells (APCs). Thus, B cells induced Th2 shifts, whereas monocytes favoured Th1 induction. Moreover, IL-12 in conditions with B cells counteracted their Th2 bias. Interestingly, in purified naïve T-cell cultures, containing a small population of HLA-DR+ T cells, Th1/Th2 differentiation can be induced by TSST-1 too. There, Th-cell polarization is strongly dependent on TSST-1 concentration, indicating that this is a key parameter in regulating the differentiation of T cells. In conclusion, our data show that Th1/Th2 differentiation of TSST-1-stimulated naïve T cells is controlled by the type of APCs, and in APC-depleted cultures, it depends on the presence of HLA-DR+ cells and TSST-1 concentration.

Inhibition of proliferation and apoptosis by the transcriptional repressor Mad1. Repression of Fas-induced caspase-8 activation.

Mad1 is a member of the Myc/Max/Mad network of transcriptional regulators that play a central role in the control of cellular behavior. Mad proteins are thought to antagonize Myc functions at least in part by repressing gene transcription. To systematically examine the function of Mad1 in growth control and during apoptosis, we have generated U2OS cell clones that express Mad1 under a tetracyline-regulatable promoter (UTA-Mad1). Mad1 was induced rapidly and efficiently, localized to the nucleus, and bound to DNA as a heterodimer with Max. The induction of Mad1 reduced cellular growth and, more profoundly, inhibited colony formation of UTA-Mad1 cells. Conditioned medium neutralized this inhibitory effect implying that Mad1 function is regulated by extracellular signals. In addition Mad1 interfered with Fas-, TRAIL-, and UV-induced apoptosis, which coincided with a reduced activation of caspase-8 during Fas-mediated apoptosis in response to Mad1 expression. Furthermore, microinjection of Mad1-expressing plasmids into fibroblasts inhibited apoptosis induced by the oncoproteins c-Myc and E1A. Thus, Mad1 not only interferes with cellular proliferation but also with apoptosis, which defines a novel aspect of Mad1 function.

Preparation of autologous platelets for the ophthalmologic treatment of macular holes.

BACKGROUND: Platelet concentrates were recently used for ophthalmologic treatment of macular holes. This strategy was investigated to define standardized blood bank components. STUDY DESIGN AND METHODS: Two different, highly concentrated autologous platelet components, one from whole-blood preparation and the other from plateletpheresis, were evaluated. In the first procedure, platelet-rich plasma was obtained from 250 mL of whole blood. After storage for 20 hours, platelet-rich plasma was concentrated in a second centrifugation step and adjusted to 10 x 10(9) platelets per mL. In the second procedure, platelets were collected by apheresis, stored overnight, centrifuged, and adjusted to 20 x 10(9) platelets per mL. The respective component was instilled during vitrectomy and gas tamponade in patients with stage II to IV macular holes. Patients were followed for 9 months. RESULTS: With regard to the various preparation procedures and final concentrations of platelets in the components, no differences in wound healing were observed. An anatomic closure of the macular hole was achieved in 18 of 19 treated patients. Visual acuity improved in 14 patients. CONCLUSION: Both types of highly concentrated platelet components were effective in achieving high closure rates of macular holes. These autologous platelet components possess the quality standard of blood bank components and could be of great benefit for initiating wound healing in other clinical settings.

A new in vitro model for studying human T cell differentiation: T(H1)/T(H2) induction following activation by superantigens.

A new T(H1)/T(H2) in vitro model for mechanistic studies and drug screening in human T cells was established working with ficoll-separated PBMCs or elutriated lymphocytes cultured in serum-free medium. Human T cells could be kept viable and reactive in this medium for several months. In this model, superantigens (SAs) were used to activate exclusively those T cell clones which were known to express specifically SA-binding Vbeta-chains of the T cell receptor. It was possible to identify the activated SA-specific T cells among the whole T cell population by using monoclonal antibodies against these Vbeta-chains and to follow responses involving receptor regulation and cytokine expression. The flow cytometric analysis revealed, that SA exposure caused an upregulation of the IL-2 receptor selectively in the SA-specific subpopulation. Only the T cells of this subpopulation could be shifted towards T(H1) or T(H2) differentiation, which was determined by the distribution of IFN-gamma and IL-4 positive cells. Regulation of IFN-gamma could be detected by flow cytometry after 18 h and that of IL-4 on the third day of cell culture. The differentiation status could be influenced by various measures: T(H1) shifts were achieved in the presence of IL-12 and anti-IL-4, whereas, T(H2) shifts were induced more slowly with monocyte-reduced elutriated lymphocytes in the presence of IL-4, IL-6, anti-IL-12, 1alpha,25-dihydroxy-vitamin-D3 or combinations thereof. It was found that sialidase stimulated whereas TGF-beta and pentoxifylline suppressed both kinds of T cell response. The T(H1)/T(H2) differentiation persisted for several weeks after primary activation if cells were expanded in IL-2 containing serum-free culture medium. Therefore, this human T(H1)/T(H2) in vitro model should be ideal for studying early and late events of infection, allergy, and autoimmunity as well as for investigating the cellular interactions involved. In addition, the early detection of the response pattern makes this model potentially useful for drug screening.

Molecular epidemiology of HIV in Israel.

The aim of this study was to identify the HIV types and subtypes prevalent in Israel among different populations in terms of risk or geographic origin of the HIV infection. A total of 149 blood samples were collected from HIV-positive persons from different risk groups for HIV infection who were living in Israel. HIV subtyping was performed by a V3-based peptide enzyme immunoassay, supplemented by direct sequencing of polymerase chain reaction products from the V3 region. Multiple HIV-1 subtypes were shown to circulate in Israel; whereas most of the infections among Israelis and Palestinians were of subtype B, infections among the large Ethiopian population in Israel were caused by HIV-1 subtype C. Occasionally, we found HIV-1 subtypes A and D and a putative B/C recombinant. No HIV-2 infection was identified. Sequence comparisons and phylogenetic tree analyses point at multiple introductions of HIV into the country. The presence of mainly two different HIV-1 subtypes, B and C, in two separated populations in Israel may result in two distinct epidemiologic patterns among HIV-infected individuals in Israel. Subtype C infection among the Ethiopians in Israel opens new research avenues toward better understanding the natural history of infection with HIV-1 subtype C in Ethiopians living in a Western society compared with those living in Ethiopia.

Secondary structural elements as a basis for antibody recognition in the immunodominant region of human immunodeficiency viruses 1 and 2.

Synthetic peptide antigens corresponding to the entire third variable region V3, the principal neutralizing determinant of the human immunodeficiency virus (HIV) envelope glycoprotein of HIV-1 subtype B (1), HIV-2 subtype A (5), and HIV-2 subtype B (7) were synthesized by solid-phase peptide synthesis (Table 1). 1 and 5 were also prepared as their GlcNAc-glycosylated forms at the natural N-glycosylation site NXT (positions 6-8; peptides 4 and 6). Additionally, the proposed beta-turn region of 1 (GPGR; positions 15-18) was altered by introducing D-Ala17 (2) and D-Pro16 (3). All compounds have been studied by two-dimensional NMR techniques. Interproton distances and 3JNH/H alpha coupling constants derived from NMR data are used as restraints in distance geometry and ENSEMBLE-Distance and angle-bound driven dynamics calculations. The stimulations led to disordered conformations except for a high propensity of a beta II-turn in the region GPXR (positions 15-18) in 1, 2, and 4. In 3 (G-D-ProGR, positions 15-18), a type beta I'-turn was mainly found instead. For peptide 7, the consensus sequence of HIV-2 subtype B, a type beta II-turn was also found although the primary structure (VSGL; positions 15-18) differs grossly from the HIV-1 peptide 1. With the exception of 2, all beta II-turns were able to form a canonically opened beta-turn by a 180 degree rotation of phi(G17). Surprisingly, compounds 5 and 6 that are highly similar to 7 showed no beta II-type turn within MSGL (positions 15-18). They form a type beta VIII-turn across the tetrapeptide SGLV (positions 16-19) together with a non-canonical turn conformation across LMSG (positions 14-17) leading to an S-conformation. The reaction of the peptides with HIV-positive sera from patients infected with different subtypes of HIV-1 and HIV-2 was tested in enzyme-linked immunosorbent assays (ELISA reactions). No HIV-2 sera reacted peptide 1 and no HIV-1 sera showed reactivity to peptide 5. We propose that certain amino acid exchanges within the V3 domain lead to altered conformations of the V3 loop resulting in antibodies that show altered binding properties to the peptide antigens used in the ELISA reactions.

The antitumor potency of progesterone antagonists is due to their differentiation potential.

A new therapy for the progesterone receptor positive mammary carcinoma may be the treatment with progesterone antagonists. This new class of antihormones causes a strong inhibition of tumor growth comparable to the potency of ovariectomy in a panel of experimental mammary carcinomas. The mechanisms of the strong tumor-inhibiting action of progesterone antagonists on experimental mammary carcinomas mainly depends on a progesterone receptor mediated process leading to induction of terminal differentiation and a blockade of the cell cycle. To further characterize the antitumor mechanism of progesterone antagonists we analyzed the effects of Onapristone and ZK 112.993 on DMBA- and MNU-mammary tumors of the rat and MXT-tumors of the mouse after different therapy intervals. These hormone-dependent mammary tumors normally display intraductal growth in papillary, cribiform or solid formation, whereas after treatment periods of 2-6 weeks with progesterone antagonists they displayed dysplastic ductal and acinous formations, usually filled with secretory material. Whereas tumor size, mitotic index, and the grade of tumor malignancy decreased distinctly, the volume fraction of glandular structures in the tumors as well as the appearance of apoptosis increased 3-fold compared to the controls. In addition, the mammary glands of progesterone antagonist treated animals showed the morphological features of differentiation with the appearance of secretory activity. Interestingly, the staining pattern of some of the lectins used, especially UEA 1 binding pattern, fits to the concept of differentiation since recent studies revealed a higher degree of fucosylation only in benign lesions of human breast cancers. Therefore, these data underline the concept of a differentiation potential of progesterone antagonists on progesterone receptor positive mammary carcinomas.