Hospital-acquired invasive group a streptococcal infections in Ontario, Canada, 1992-2000.

BACKGROUND: A significant proportion of invasive group A streptococcal infections are hospital acquired. No large, prospective studies have characterized this subgroup of cases and evaluated the risk of transmission in hospitals. METHODS: We conducted prospective, population-based surveillance of invasive group A streptococcal infections in Ontario, Canada, from 1992 to 2000. Epidemiologic and microbiologic investigations were conducted to identify cross-transmission. RESULTS: We identified 291 hospital-acquired cases (12.4%) among 2351 cases of invasive group A streptococcal disease. Hospital-acquired invasive group A streptococcal infections are heterogeneous, including surgical site (96 cases), postpartum (86 cases), and nonsurgical, nonobstetrical infections (109 cases). Surgical site infections affected 1 of 100,000 surgical procedures and involved all organ systems. Postpartum infections occurred at a rate of 0.7 cases per 10,000 live births and exhibited an excellent prognosis. Nonsurgical, nonobstetrical infections encompassed a broad range of infectious syndromes (case-fatality rate, 37%). Nine percent of cases were associated with in-hospital transmission. Transmission occurred from 3 of 142 patients with community-acquired cases of necrotizing fasciitis requiring intensive care unit (ICU) admission, compared with 1 of 367 patients with community-acquired cases without necrotizing fasciitis admitted to the ICU and 1 of 1551 patients with other cases (P<.001). Fifteen outbreaks were identified; 9 (60%) involved only 2 cases. Hospital staff were infected in 1 of 15 outbreaks, but colonized staff were identified in 6 (60%) of 10 investigations in which staff were screened. CONCLUSIONS: Presentation of hospital-associated invasive group A streptococcal infections is diverse. Cross-transmission is common; illness occurs in patients but rarely in staff. Isolation of new cases of necrotizing fasciitis and intervention after a single nosocomial case may also prevent transmission.

Apoptosis regulation in the mammary gland.

Epithelial apoptosis has a key role in the development and function of the mammary gland. It is involved with the formation of ducts during puberty and is required to remove excess epithelial cells after lactation so that the gland can be prepared for future pregnancies. Deregulated apoptosis contributes to malignant progression in the genesis of breast cancer. Since epithelial cell apoptosis in the lactating mammary gland can be synchronised by forced weaning, it has been possible to undertake biochemical analysis of the pathways involved. Together with the targeted overexpression or deletion of candidate genes, these approaches have provided a unique insight into the complex mechanisms of apoptosis regulation in vivo. This review explores what is currently known about the triggers for apoptosis in the normal mammary gland, and how they link with the intrinsic apoptotic machinery.

Characterization of the CD154-positive and CD40-positive cellular subsets required for pathogenesis in retrovirus-induced murine immunodeficiency.

Genetically susceptible C57BL/6 (B6) mice that are infected with the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell lymphomas, and an immunodeficiency state bearing many similarities to the pathologies seen in AIDS. Because of these similarities, this syndrome has been called murine AIDS (MAIDS). We have previously shown that CD154 (CD40 ligand)-CD40 molecular interactions are required both for the initiation and progression of MAIDS. Thus, in vivo anti-CD154 monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in LP-BM5-infected wild-type mice when either a short course of anti-CD154 MAb treatment was started on the day of infection or a course was initiated 3 to 4 weeks after LP-BM5 administration, after disease was established. Here, we further characterize this required CD154-CD40 interaction by a series of adoptive transfer experiments designed to elucidate which cellular subsets must express CD154 or CD40 for LP-BM5 to induce MAIDS. Specifically with regard to CD154 expression, MAIDS-insusceptible B6 nude mice reconstituted with highly purified CD4+ T cells from wild-type, but not from CD154 knockout, B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast, nude B6 recipients that received CD8+ T cells from wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6 CD40 knockout mice, which are also relatively resistant to LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease after reconstitution with highly purified wild-type B cells but not after receiving purified wild-type dendritic cells (DC) or a combined CD40+ population composed of DC and macrophages obtained from B6 SCID mouse donors. Based on these and other experiments, we thus conclude that the cellular basis for the requirement for CD154-CD40 interactions for MAIDS induction and progression can be accounted for by CD154 expression on CD4+ T cells and CD40 expression on B cells.

Location of a ligand recognition site of FMRFamide-gated Na(+) channels.

The second FMRFamide-gated Na(+) channel (HtFaNaC), from Helisoma trivolvis, has been cloned. HtFaNaC has some different pharmacological properties to HaFaNaC, from Helix aspersa, which has enabled a rational approach to be made to start to identify the FMRFamide recognition site. Several chimeras were made by switching sections between the channels. The differences in sensitivity to FMRFamide, and amiloride, were assessed after expression in Xenopus oocytes. The data suggest that a recognition site for FMRFamide, and the potentiating action of amiloride, resides in a sequence of about 120 amino acids in the extracellular loop proximal to the first transmembrane segment.

Cloning and expression of a FMRFamide-gated Na(+) channel from Helisoma trivolvis and comparison with the native neuronal channel.

We have cloned a cDNA encoding a Phe-Met-Arg-Phe-NH(2) (FMRFamide)-gated Na(+) channel from nervous tissue of the pond snail Helisoma trivolvis (HtFaNaC) and expressed the channel in Xenopus oocytes. The deduced amino acid sequence of the protein expressed by HtFaNaC is 65 % identical to that of the FMRFamide-gated channel cloned from Helix aspersa (HaFaNaC). HtFaNaC expressed in oocytes was less sensitive to FMRFamide (EC(50) = 70 microM) than HaFaNaC (EC(50) = 2 microM). The two had a similar selectivity for Na+. The amplitude of the FMRFamide response of HtFaNaC was increased by reducing the extracellular concentration of divalent cations. The conductance of the two channels was similar, but the mean open time of unitary events was shorter for expressed HtFaNaC compared to expressed HaFaNaC. Each channel was susceptible to peptide block by high agonist concentrations. In marked contrast to HaFaNaC and other amiloride-sensitive Na(+) channels, amiloride, and the related drugs benzamil and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), enhanced the FMRFamide response in oocytes expressing HtFaNaC cRNA. The potentiating effects of EIPA and benzamil were greater than those of amiloride. Unitary current analysis showed that with such drugs, there was channel blockade as well as an increased probability of channel opening. The similar permeability of the oocyte-expressed HtFaNaC and the Helisoma neuronal channel, and the susceptibility of both to agonist blockade and blockade by divalent cations, suggest that the channels are the same. However, neuronal channels were less susceptible to enhancement by amiloride analogues and in some patches were more sensitive to FMRFamide than expressed HtFaNaC.

Immune CD8(+) T cells prevent reactivation of Toxoplasma gondii infection in the immunocompromised host.

Toxoplasma gondii remains a serious cause of morbidity and mortality in individuals that are immunosuppressed, patients with AIDS in particular. The cellular immune response, especially by gamma interferon (IFN-gamma)-producing CD8(+) T cells, is an essential component of protective immunity against the parasite. In the present study the role of CD8(+) T cells during the reactivation of Toxoplasma infection in an immunocompromised murine model was evaluated. Chronically infected mice were challenged with LP-BM5 virus, and the kinetics of CD8(+) T-cell function was studied. At 10 weeks after viral infection, mice showed obvious signs of systemic illness and began to die. At this stage, CD8(+) T cells were unresponsive to antigenic stimulation and unable to kill Toxoplasma-infected targets. IFN-gamma production by the CD8(+) T cells from dual-infected animals reached background levels, and a dramatic fall in the frequency of precursor cytotoxic T lymphocytes was observed. Histopathological analysis of the tissues demonstrated signs of disseminated toxoplasmosis as a result of reactivation of infection. However, treatment of the dual-infected animals with immune CD8(+) T cells at 5 weeks post-LP-BM5 challenge prevented the reactivation of toxoplasmosis, and mice continued to live. Our study for the first time demonstrates a therapeutic role for CD8(+) T cells against an opportunistic infection in an immunocompromised state.

Block of the helix FMRFamide-gated Na+ channel by FMRFamide and its analogues.

1. In Helix neurones high doses of Phe-Met-Arg-Phe-NH2 (FMRFamide) often evoke biphasic inward whole-cell currents with brief application, and suppression of the current with prolonged application. With outside-out patches, a transient early suppression of the unitary current amplitude was seen following application of high doses of FMRFamide. 2. Continuous application of a concentration of FMRFamide from 30 microM to 1 mM resulted in a reduction in the amplitude of the unitary currents and an increase in open state noise. There was also an increase in the occurrence of smaller, 'subconductance' currents with the higher concentrations of FMRFamide. Similar effects were seen with FMRFamide on FaNaC expressed in oocytes. The FMRFamide analogues FLRFamide and WnLRFamide were more effective in evoking the lower conductance state. These effects of agonist at high concentrations were voltage dependent suggesting channel block. 3. A similar effect was seen when one of the related peptides FKRFamide, FM(D)RFamide, nLRFamide or N-AcFnLRFamide was co-applied with a low FMRFamide concentration. However, the non-amidated peptides FKRF, FLRF and nLRF and also WMDFamide did not have this effect. 4. The inhibition of unitary currents induced by amiloride was qualitatively different from that produced by FMRFamide analogues with no obvious occurrence of subconductance levels. FMRFamide-gated channels were also blocked by guanidinium, but only at very high concentrations. 5. The results strongly suggest a partial inhibition of current flow through the FMRFamide- gated channel by some part of the agonist or the related antagonist peptide molecules.

Leptospirosis associated with severe pulmonary haemorrhage in Far North Queensland.

Pulmonary haemorrhage as a manifestation of leptospirosis is rarely diagnosed in developed countries. Five patients with proven leptospirosis associated with severe pulmonary haemorrhage presented to one hospital in Far North Queensland between January 1994 and June 1997. Four required admission to the intensive care unit and one patient died. Pulmonary haemorrhage is an uncommon but severe complication of leptospirosis and may be a source of diagnostic confusion in tropical areas of Australia.

Evidence for a continued requirement for CD40/CD40 ligand (CD154) interactions in the progression of LP-BM5 retrovirus-induced murine AIDS.

In genetically susceptible C57BL/6 mice the LP-BM5 isolate of murine retroviruses causes profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, and an immunodeficiency syndrome bearing many similarities to the pathologies seen in AIDS. Because of these similarities, which also include terminal B cell lymphoma formation, this syndrome has been called murine AIDS or MAIDS. Prompted by previous reports showing that the onset of MAIDS is dependent on the presence of both CD4+ T and B cells, we have previously shown that anti-gp39/CD40 ligand mAb (anti-CD40L mAb) treatment of LP-BM5-infected mice is effective in inhibiting the induction of MAIDS when a short course of anti-CD40L mAb treatment was started on the same day as LP-BM5 administration. The success of anti-CD40L mAb therapy, as indicated by a much reduced degree of splenomegaly, hypergammaglobulinemia, and mitogen and allogeneic CTL unresponsiveness, demonstrated that CD40L/CD40 interactions were critical to the establishment of MAIDS. Here we extend these findings through the use of delayed anti-CD40L mAb treatment of mice, beginning 3-4 weeks after LP-BM5 infection, by showing that interruption of CD40L/CD40 interactions also interferes with the progression of MAIDS. About 60% of LP-BM5-preinfected mice were affected by delayed anti-CD40L mAb treatment, with substantially reduced spleen weights and serum hypergammaglobulinemia and normal or greatly restored proliferative responses to Con A stimulation and CTL responses to allogeneic stimulation. The other LP-BM5-infected mice that did not respond to anti-CD40L therapy were found to have made antibodies to the anti-CD40L mAb. Thus, in a majority of mice anti-CD40L mAb therapy was very effective in interfering with MAIDS pathogenesis well after the establishment of the virus infection and MAIDS symptomatology, indicating that CD40L/CD40 interactions are crucial to the maintenance and progression of the disease, as well as its initiation.

Body composition, energy expenditure, and energy intake in patients with Williams syndrome.

OBJECTIVE: To evaluate the body composition, resting energy expenditure (REE), and energy intake of adolescents and adults with Williams syndrome (WS) compared with matched healthy control subjects. METHODS: Body composition was determined by total body electrical conductivity and anthropometric measurements in six subjects with WS from the WS Clinic at Children's Hospital of Philadelphia and six healthy control subjects matched for age, height, and pubertal stage. REE was measured by open-circuit indirect calorimetry. Dietary intake was assessed by 3-day dietary records. RESULTS: Subjects with WS had similar anthropometric measurements to the control group except for a significantly lower percent body fat (17.1%+/-5.2% vs. 25.0%+/-6.7%). Dietary intake (measured in kilocalories per day) was similar between the two groups. REE was statistically higher by 155 kcal/day in the WS group after controlling for age, gender, and body composition. In addition, the WS group had a significantly higher percent predicted REE according to the World Health Organization equation, which adjusts for age, gender, and body weight. CONCLUSION: Adolescents and adults with WS have a similar dietary intake but a lower body fat than healthy control subjects. A higher REE may contribute to the thin body habitus and reduced total body fat stores of people with WS.

Modulation of ligand-gated dopamine channels in Helix neurones.

Dopamine gates a fast excitatory response in Helix C2 neurones. Whole cell, and multiple unitary dopamine-gated currents showed variable decay rates and desensitization properties, suggesting the presence of more than one channel type. Manipulation of internal free [Ca2+] by various procedures (external zero Ca2+ or 1 mM Co2+, prolonged depolarization, A23187, or flufenamic acid), affected both the amplitude and decay time for the response, and also suggested the presence of separate fast and slowly decaying components. Responses were prolonged by intracellular fluoride a non specific phosphatase inhibitor, and attenuated and shortened by the protein kinase inhibitors H7 and staurosporine, and the calmodulin inhibitor W7. Phorbol ester potentiated and prolonged the response and this effect was reversibly antagonized by the specific protein kinase C inhibitor chelerythrine. Different dopamine-activated unitary currents were distinguished in outside-out patches by conductance (5, 8, 12 and 15pS), rate of recovery from desensitization, and pattern of openings. Discrimination of slow and fast components of the response was possible with apomorphine, ADTN, and caffeine. Paradoxically the dopamine antagonists chlorpromazine and spiperone, but not dopamine itself, stimulated sustained activity of 5pS unitary currents which did not desensitize in outside-out patches. Modulation of different channels underlying the fast dopamine response by protein kinase C, and possibly other mechanisms, provides a potent means of controlling excitatory dopaminergic synaptic transmission.

Ligand-gated ion channels opened by 5-HT in molluscan neurones.

1. 5-Hydroxytryptamine (5-HT) activated a fast (70 ms to half maximum) and desensitizing inward current through non-selective channels conducting predominantly monovalent cations in neurons of Helix aspersa. 2. alpha-Methyl-5-HT was equipotent with 5-HT in activating this current, but the known selective agonists at vertebrate 5-HT3 receptors, 2-methyl-5-HT and arylbiguanides were ineffective (< 100 microM). 5-Methoxytryptamine which is inactive on vertebrate 5-HT3 receptors was a very weak agonist. 3. The responses were antagonized by the specific vertebrate 5-HT3 receptor blocker MDL-72222 (IC50 = 1 microM), but were only weakly affected by ondansetron (10 microM). The 5-HT2-type antagonist, ketanserin (< 5 microM) had no effect. The responses were also antagonized by the non-specific antagonists (+)-tubocurarine and strychnine. 4. Unitary currents through channels non-selective for monovalent cations, and with a conductance of 2pS, could be activated repeatedly by 5-HT or alpha-methyl-5-HT in outside-out patches from neurones exhibiting the fast 5-HT-activated current (I[5-HT]fast), even in the presence of 500 microM GDP-[beta S] in the recording pipette. This strongly supports direct-gating of these channels by 5-HT. The properties of these unitary currents resembled those of I[5-HT]fast. 5. The pharmacological properties of this molluscan 5-HT-operated, ligand-gated channel differed sufficiently from known vertebrate 5-HT3-type receptors to suggest that it represents a new class of 5-HT receptor.

Antibody to the ligand for CD40 (gp39) inhibits murine AIDS-associated splenomegaly, hypergammaglobulinemia, and immunodeficiency in disease-susceptible C57BL/6 mice.

Infection of genetically susceptible C57BL/6 mice with the LP-BM5 isolate of murine retroviruses cause profound splenomegaly, hypergammaglobulinemia, lymphadenopathy, and an immunodeficiency syndrome which includes the development of terminal B-cell lymphomas. Because many of these and the other manifestations of LP-BM5 virus-induced disease are similar to those seen in AIDS, this syndrome has been named murine AIDS, or MAIDS. Previous reports have shown that the onset of MAIDS depends on the presence of both CD4+ T cells and B cells and have suggested that CD4+ T-cell-B-cell interactions are important to disease pathogenesis. Here, we assessed the possibility that interactions between CD40 and its ligand on activated CD4+ T cells, CD40 ligand/gp39, are involved in the development of MAIDS. To test this hypothesis, LP-BM5-infected B6 mice were treated in vivo with anti-gp39 monoclonal antibody. As a result, MAIDS-associated splenomegaly, hypergammaglobulinemia, germinal center formation, and the loss of in vitro responsiveness to the T- and B-cell mitogens concanavalin A and lipopolysaccharide were inhibited. Anti-gp39 monoclonal antibody-treated LP-BM5-infected mice were also able to mount essentially normal alloantigen-specific cytolytic T-lymphocyte responses. These results support the possibility that molecular interactions between CD40 and gp39 are critical to the development of MAIDS.

Dopamine directly activates a ligand-gated channel in snail neurones.

Synaptically released dopamine is known to evoke fast as well as slow synaptic potentials in neurones of gastropod molluscs. Here evidence is presented that the fast excitatory response to dopamine is mediated by the direct activation of a ligand-gated channel: unitary currents were observed in outside-out patches of neurones exposed to dopamine, and the response persisted in the presence of intracellular guanosine 5'-o-(2-thiobiphosphate), GDP[beta-S], a condition known to block G-protein-coupled responses to dopamine and other agents. In whole-cell recordings, the fast response desensitized very rapidly; it was less desensitized in outside-out patches, suggesting dependence of desensitization on an intracellular factor. The response to dopamine was blocked by D-tubocurarine and strychnine (both probably by channel blockade), by apomorphine, chlorpromazine and relatively high doses of (+/-)-sulpiride and spiperone. The channel conducts predominantly monovalent cations. Unexpectedly, the fast response to dopamine was also observed in an identified dopaminergic neurone when maintained in isolation in culture. The receptors on the dopaminergic neurone were unevenly distributed, being more abundant on the axon-hillock, axon and neurite terminals.

Gadolinium-based MR contrast media: potential for growth of microbial contaminants when single vials are used for multiple patients.

OBJECTIVE: To assess potential risk if single-dose vials of gadolinium-based contrast media are used for multiple patients. The use of multidose vials can pose a significant risk if contaminants are accidentally introduced into the vials and proliferate. Therefore, we tested the ability of gadolinium-based contrast to support the growth of a variety of microbial pathogens. MATERIALS AND METHODS: We collected the unused portions of 15 single-use vials of gadolinium-based contrast media. The residual contrast material was refrigerated after use and checked for sterility prior to inoculation with a selection of gram-positive and gram-negative bacteria and yeast. Contrast material was incubated at room temperature and at 4 degrees C, and quantitative cultures were performed at 0, 24, 48, and 72 hr and at 7 days. RESULTS: No microbial growth occurred from cultures of the used contrast solutions prior to inoculation. None of the organisms tested in our study proliferated in the contrast material. All test organisms persisted at least 48 hr after inoculation at both temperatures. At 7 days, Staphylococcus aureus, Streptococcus epidermidis, and Corynebacterium jeikeium were recovered in significant quantities. Colony counts of Serratia odorifera rapidly decreased at room temperature but persisted beyond 7 days at 4 degrees C. CONCLUSION: The risk of contaminating vials of contrast agents punctured aseptically is small. Our study demonstrates a lack of proliferation of organisms in the two gadolinium-based compounds tested, suggesting that these solutions could be used for more than one procedure.

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) directly gates two ion channels in an identified Helix neurone.

FMRFamide (i.e. Phe-Met-Arg-Phe-NH2) application to the C2 neurone of Helix caused a depolarizing response which consisted of a large, rapidly developing, and rapidly desensitizing inward current, underlain by a smaller, slower inward current which did not desensitize. Both currents were carried through sodium-selective channels which were insensitive to D-tubocurarine, and the to the fast sodium channel blockers tetrodotoxin (TTX) and lignocaine. Only the faster, desensitizing current could be blocked by amiloride. FMRFamide also activated two types of unitary inward currents with slightly differing amplitudes in outside-out patches taken from the C2 neurone, both through sodium-selective ion channels. Only the smaller unitary currents readily desensitized and were susceptible to block by amiloride, and they also activated more rapidly. Unitary currents of both types were recorded in outside-out patches in the absence of freely diffusible intracellular mediators, and were also activated when guanosine 5'-O-(2-thiodiphosphate) (GDP [beta-S]) was included in the recording pipette solution. This supports a tight receptor/channel coupling for both responses, with no involvement of GTP-binding proteins. Further, the very fast rate of activation of the smaller channels, which generally carry the major part of the FMRFamide-induced current, strongly indicates that these channels are ligand gated.

Adoptive transfer of polyclonal and cloned cytolytic T lymphocytes (CTL) specific for mouse AIDS-associated tumors is effective in preserving CTL responses: a measure of protection against LP-BM5 retrovirus-induced immunodeficiency.

Cytolytic T lymphocytes (CTL) can be raised against C57BL/6 B-cell lymphomas from mice with LP-BM5 murine leukemia virus-induced AIDS (MAIDS). Adoptive transfer of polyclonal anti-MAIDS tumor CTL or two CTL clones specific for the B6-1710 MAIDS lymphoma caused preservation of major histocompatibility complex-restricted and allogeneic CTL responses, which may be interpreted as indices of protection from LP-BM5 murine leukemia virus-induced immunodeficiency.

Is a low cerebrospinal fluid blood glucose ratio indicative of infection in patients with post haemorrhagic hydrocephalus?

Cerebrospinal fluid glucose and cerebrospinal fluid:blood glucose ratios were compared in seven patients with post haemorrhagic hydrocephalus having lumbar puncture/ventricular tap as a therapeutic measure. A control group of 10 babies was used, without intraventricular haemorrhage, and having lumbar puncture as part of a septic screen. Of 50 separate taps in the patient group, 38% had blood glucose measured and 76% had CSF glucose measured. Median cerebrospinal fluid glucose was 1.2 mmol (range, 0.4-2.5 mmol/l) in the patient group and 3.1 mmol/l (range, 1.4-10.3 mmol/l) in the control group. The median cerebrospinal fluid:blood glucose ratio in the patient group was 0.235 (range, 0.07-0.53) and in the control group was 0.709 (range, 0.6-1.4). Hypoglycorrhachia appears to be a normal finding in patients with post haemorrhagic hydrocephalus and does not indicate infection in these infants. Measurement of cerebrospinal fluid:blood glucose ratio is not warranted when cerebrospinal fluid is drained purely as a therapeutic measure in these patients.

Cytotoxic T lymphocytes directed against MAIDS-associated tumors and cells from mice infected by the LP-BM5 MAIDS defective retrovirus.

The LP-BM5 retrovirus, a complex containing ecotropic helper, recombinant MCF, and defective retroviruses, causes an immunodeficiency-termed mouse AIDS (MAIDS). Many disease features of MAIDS resemble those of AIDS, including terminal B cell lymphomas. Previously we generated from MAIDS-susceptible C57BL/6 mice cytolytic T lymphocytes (CTL) specific for MAIDS-associated B cell lymphomas. Data of the present study (1) exclude MCF and establish a role for defective virus in generating C57BL/6 CTL to MAIDS-associated tumors by experiments involving in vitro stimulation with cells from LP-BM5, ecotropic, or ecotropic-rescued defective virus-infected mice and (2) confirm that such CTL are specific for tumors of MAIDS origin. Several approaches testing for direct involvement of defective virus or its gag-encoded polyprotein, however, did not provide evidence that MAIDS tumor-specific CTL were directed to structural virion proteins, suggesting the possibility that such CTL are specific for nonvirion antigens whose expression depends on the action of the defective genome in the MAIDS disease process.