Genetic control of natural killing and in vivo tumor elimination by the Chok locus.

The molecular mechanisms underlying target recognition during natural killing are not well understood. One approach to dissect the complexities of natural killer (NK) cell recognition is through exploitation of genetic differences among inbred mouse strains. In this study, we determined that interleukin 2-activated BALB/c-derived NK cells could not lyse Chinese hamster ovary (CHO) cells as efficiently as C57BL/6-derived NK cells, despite equivalent capacity to kill other targets. This strain-determined difference was also exhibited by freshly isolated NK cells, and was determined to be independent of host major histocompatibility haplotype. Furthermore, CHO killing did not correlate with expression of NK1.1 or 2B4 activation molecules. Genetic mapping studies revealed linkage between the locus influencing CHO killing, termed Chok, and loci encoded within the NK gene complex (NKC), suggesting that Chok encodes an NK cell receptor specific for CHO cells. In vivo assays recapitulated the in vitro data, and both studies determined that Chok regulates an NK perforin-dependent cytotoxic process. These results may have implications for the role of NK cells in xenograft rejection. Our genetic analysis suggests Chok is a single locus that affects NK cell-mediated cytotoxicity similar to other NKC loci that also regulate the complex activity of NK cells.

The Ly-49D receptor activates murine natural killer cells.

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Nonstochastic coexpression of activation receptors on murine natural killer cells.

Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti-Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3(+) T cells, indicating that Ly-49H may be an NK cell-specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1(+) cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.

Autoimmunity and increased c-myb transcription.

A single recessive gene, lpr, induces an autoimmune-lymphoproliferative syndrome in several strains of mice. The lymphoid organs of lpr/lpr mice contained cells with increased amounts of myb RNA, which codes for a protein found in the nucleus. A similar human lymphoproliferative disorder also had an increase in c-myb expression. Mouse T cells induced by mitogens to proliferate did not express large amounts of myb RNA, indicating that marked myb expression is not a general feature of lymphocyte activation and proliferation.

Vital involvement of a natural killer cell activation receptor in resistance to viral infection.

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

Predicting Amputation and Multiple Debridements in Tooth Knuckle Injuries.

BACKGROUND: Tooth knuckle injuries can be expensive to treat and may necessitate amputation in some cases. Several limitations exist in the literature regarding our knowledge around the factors predicting amputation and the need for multiple debridements in treating this injury. METHODS: A historic cohort study of 321 patients treated for tooth knuckle injuries was undertaken. Twenty-one demographic, clinical and laboratory variables were collected. Two outcome measurements were collected - the need for amputation and the need for more than one surgical debridement. A multivariate logistic regression was performed to determine the relationship between the predictor variables and the outcome measurements. RESULTS: Of the 321 patients examined, 1.6% required amputations and 25% required multiple debridements. Osteomyelitis was found to be a major predictor for amputation in these patients (OR = 35). Delayed presentation (OR = 1.1) and diabetes (OR = 2.6) were found to significantly increase the risk of requiring multiple debridements. CONCLUSIONS: Our models were able to predict what patients were at the greatest risk for amputation and multiple debridement. Reducing rates of osteomyelitis and delays in presentation may help reduce the incidence of amputation and reoperation in this injury.

Studies of the pathogenesis of angioimmunoblastic lymphadenopathy.

We studied the immune functions of two patients with angioimmunoblastic lymphadenopathy (AILD) in an attempt to determine whether the B cells were primarily hyperactive or, rather, if T cell abnormalities might underlie the B cell hyperactivity observed in these patients. We found that the B cells of the AILD patients did not proliferate spontaneously, nor were they induced to proliferate excessively by fresh normal T cells. In contrast, AILD T cells induced both autologous and allogeneic B cells to proliferate and to differentiate into Ig secreting cells. Spontaneous culture supernates of T cells obtained from each patient induced substantial proliferation of B cells (B cell-activating activity) as well as proliferation in a standard costimulatory assay (B cell growth factor activity). The culture supernate of a T cell line, which was established from one patient, showed both activities. The T cell line supernate also induced Ig production by staphylococcal A Cowan-activated B cells. None of these properties of AILD T cells was found among 10 normal controls. The addition of AILD T cells to autologous or allogeneic B cells in the presence of pokeweed mitogen (PWM) led to marked suppression of both proliferation and Ig production. This was true even in the presence of fresh normal T cells. Pretreatment studies showed that suppressor cells were induced by the interaction of AILD T cells with PWM-activated B cells. The present study suggests that the B cell hyperactivity observed in AILD patients might in part be due to excessive T cell effects on B cells. In addition, our results may help clarify the paradoxical impaired responsiveness to in vitro stimulation with PWM by active B cells from patients with autoimmune diseases.

Clonality of angioimmunoblastic lymphadenopathy and implications for its evolution to malignant lymphoma.

To investigate the relationship of the lymphoid hyperplasia of angioimmunoblastic lymphadenopathy with dysproteinemia (AILD) to supervening malignant lymphoma, we subjected DNA from lymph nodes and peripheral blood mononuclear cells from five AILD patients to Southern blot analysis to detect clonal rearrangements of immunoglobulin and T-cell receptor genes. Lymph nodes and peripheral blood from AILD patients were found to contain clones of lymphoid cells harboring either immunoglobulin or T-cell receptor gene rearrangements that, in some instances, regressed during the course of disease. A lymph node from one patient was involved by immunoblastic lymphoma and manifested an additional gene rearrangement pattern not seen in premalignant specimens from that patient. In contrast, DNA obtained from normal peripheral blood mononuclear cells and 11 examples of other forms of lymphoid hyperplasia showed no gene rearrangements. As a disorder of cellular immunoregulation in which lymphoid cells may escape normal growth controls, AILD provides a natural model to dissect stages of lymphomagenesis in man.

Distribution of norepinephrine transporters in the non-human primate brain.

Noradrenergic terminals in the central nervous system are widespread; as such this system plays a role in varying functions such as stress responses, sympathetic regulation, attention, and memory processing, and its dysregulation has been linked to several pathologies. In particular, the norepinephrine transporter is a target in the brain of many therapeutic and abused drugs. We used the selective ligand [(3)H]nisoxetine, therefore, to describe autoradiographically the normal regional distribution of the norepinephrine transporter in the non-human primate central nervous system, thereby providing a baseline to which alterations due to pathological conditions can be compared. The norepinephrine transporter in the monkey brain was distributed heterogeneously, with highest levels occurring in the locus coeruleus complex and raphe nuclei, and moderate binding density in the hypothalamus, midline thalamic nuclei, bed nucleus of the stria terminalis, central nucleus of the amygdala, and brainstem nuclei such as the dorsal motor nucleus of the vagus and nucleus of the solitary tract. Low levels of binding to the norepinephrine transporter were measured in basolateral amygdala and cortical, hippocampal, and striatal regions. The distribution of the norepinephrine transporter in the non-human primate brain was comparable overall to that described in other species, however disparities exist between the rodent and the monkey in brain regions that play a role in such critical processes as memory and learning. The differences in such areas point to the possibility of important functional differences in noradrenergic information processing across species, and suggest the use of caution in applying findings made in the rodent to the human condition.

Predicting serious complications and high cost of treatment of tooth-knuckle injuries: a systematic literature review.

PURPOSE: The tooth-knuckle injury (TKI) is a serious and potentially costly injury seen in orthopaedic practice. The aim was to conduct a systematic literature review on the factors associated with serious complications and high treatment costs in tooth-knuckle injuries. METHODS: MEDLINE, Scopus and CINAHL were used as the literature sources. INCLUSION CRITERIA: Original research papers that reported on factors predicting serious complications and high treatment costs in TKIs were included. There were no restrictions placed on study size, language, study design or date of publication. EXCLUSION CRITERIA: Case studies, case series and review articles were not included. RESULTS: After duplicates were removed, 403 unique studies remained; after titles and abstracts were screened, 48 titles remained and were retrieved in full text. Of these, 14 titles met the inclusion criteria and were included in the data synthesis. Tenosynovitis, septic arthritis, osteomyelitis and residual stiffness were common serious complications occurring in up to 36.3, 70.0, 47.6 and 65.3 % of cases, respectively. Amputation was also common in up to 18.0 % of injuries. Treatment costs were measured by length of hospital stay and the number of debridements required. On average, patients required 3.8-8 days of admission and 1.3-2.7 debridements each. CONCLUSION: Increased time delay from injury to treatment, deeply penetrating injuries, proximal interphalangeal joint (PIPJ) injuries and, possibly, E. corrodens infections were associated with serious complications in TKIs. Delayed treatment, inadequate treatment, PIPJ injuries and deeply penetrating injuries predicted higher treatment costs. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42016029949 ( http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42016029949 ).

Progression of changes in dopamine transporter binding site density as a result of cocaine self-administration in rhesus monkeys.

The present study examined the time course of alterations in levels of dopamine transporter (DAT) binding sites that accompany cocaine self-administration using quantitative in vitro receptor autoradiography with [(3)H]WIN 35,428. The density of dopamine transporter binding sites in the striatum of rhesus monkeys with 5 d, 3.3 months, or 1.5 years of cocaine self-administration experience was compared with DAT levels in cocaine-naive control monkeys. Animals in the long-term (1.5 years) exposure group self-administered cocaine at 0.03 mg/kg per injection, whereas the initial (5 d) and chronic (3.3 months) treatment groups were each divided into lower dose (0.03 mg/kg per injection) and higher dose (0.3 mg/kg per injection) groups. Initial cocaine exposure led to moderate decreases in [(3)H]WIN 35,428 binding sites, with significant changes in the dorsolateral caudate (-25%) and central putamen (-19%) at the lower dose. Longer exposure, in contrast, resulted in elevated levels of striatal binding sites. The increases were most pronounced in the ventral striatum at the level of the nucleus accumbens shell. At the lower dose of the chronic phase, for example, significant increases of 21-42% were measured at the caudal level of the ventral caudate, ventral putamen, olfactory tubercle, and accumbens core and shell. Systematic variation of cocaine dose and drug exposure time demonstrated the importance of these factors in determining the intensity of increased DAT levels. With self-administration of higher doses especially, increases were more intense and included dorsal portions of the striatum so that every region at the caudal level exhibited a significant increase in DAT binding sites (20-54%). The similarity of these findings to previous studies in human cocaine addicts strongly suggest that the increased density of dopamine transporters observed in studies of human drug abusers are the result of the neurobiological effects of cocaine, ruling out confounds such as polydrug abuse, preexisting differences in DAT levels, or comorbid psychiatric conditions.

Orexin-saporin lesions of the medial septum impair spatial memory.

The medial septum and diagonal band of Broca (MSDB) provide a major input to the hippocampus and are important for spatial learning and memory. Although electrolytic MSDB lesions have prominent memory impairing effects, selective lesions of either cholinergic or GABAergic MSDB neurons do not or only mildly impair spatial memory. MSDB neurons are targets of orexin-containing neurons from the hypothalamus. At present, the functional significance of orexin afferents to MSDB is unclear, and the present study investigated a possible involvement of orexin innervation of the MSDB in spatial memory. Orexin-saporin, a toxin that damages neurons containing the hypocretin-2 receptor, was administered into the MSDB of rats. Rats were subsequently tested on a water maze to assess spatial reference memory and a plus maze to assess spatial working memory. At 100 ng/microl, orexin-saporin destroyed primarily GABAergic septohippocampal neurons, sparing the majority of cholinergic neurons. At 200 ng/microl, orexin-saporin almost totally eliminated GABAergic septohippocampal neurons and destroyed many cholinergic neurons. Spatial reference memory was impaired at both concentrations of orexin-saporin with a dramatic impairment observed for 24-h retention. Short-term reference memory was also impaired at both concentrations. Rats treated with 200 ng/microl, but not 100 ng/microl, of orexin-saporin were also impaired on a spontaneous alternation task, showing a deficit in spatial working memory. Our results, together with previous studies, suggest that orexin innervation of the MSDB may modulate spatial memory by acting on both GABAergic and cholinergic septohippocampal neurons.

Functional and anatomical localization of mu opioid receptors in the striatum, amygdala, and extended amygdala of the nonhuman primate.

The subregional distribution of mu opioid receptors and corresponding G-protein activation were examined in the striatum, amygdala, and extended amygdala of cynomolgus monkeys. The topography of mu binding sites was defined using autoradiography with [(3)H]DAMGO, a selective mu ligand. In adjacent sections, the distribution of receptor-activated G proteins was identified with DAMGO-stimulated guanylyl 5'(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding. Within the striatum, the distribution of [(3)H]DAMGO binding sites was characterized by a distinct dorsal-ventral gradient with a higher concentration of binding sites at more rostral levels of the striatum. [(3)H]DAMGO binding was further distinguished by the presence of patch-like aggregations within the caudate, as well as smaller areas of very dense receptor binding sites, previously identified in human striatum as neurochemically unique domains of the accumbens and putamen (NUDAPs). The amygdala contained the highest concentration of [(3)H]DAMGO binding sites measured in this study, with the densest levels of binding noted within the basal, accessory basal, paralaminar, and medial nuclei. In the striatum and amygdala, the distribution of DAMGO-stimulated G-protein activation largely corresponded with the distribution of mu binding sites. The central and medial nuclei of the amygdala, however, were notable exceptions. Whereas the concentration of [(3)H]DAMGO binding sites in the central nucleus of the amygdala was very low, the concentration of DAMGO-stimulated G-protein activation in this nucleus, as measured with [(35)S]GTPgammaS binding, was relatively high compared to other portions of the amygdala containing much higher concentrations of [(3)H]DAMGO binding sites. The converse was true in the medial nucleus, where high concentrations of binding sites were associated with lower levels of DAMGO-stimulated G-protein activation. Finally, [(3)H]DAMGO and [(35)S]GTPgammaS binding within the amygdala, particularly the medial nucleus, formed a continuum with the substantia innominata and bed nucleus of the stria terminalis, supporting the concept of the extended amygdala in primates.

Dietary copper deficiency and autoimmunity in the NZB mouse.

NZB mice were exposed from birth to a diet either adequate or deficient in copper. By age 6 wk the mice exposed to the copper-deficient diet showed symptoms characteristic of copper deficiency (anemia, hypoceruloplasminemia, and achromatrichia). The splenic lymphocytes from the copper-deficient group had reduced numbers of cells expressing the following surface markers: Ly-5, Ly-1, B-220, and sIg. Less than 10% of the splenic lymphocytes in this group were cycling, as determined by flow cytometry analysis. The spontaneous 96-h anti-ss-DNA levels in the copper-deficient group were lower than those in the control group. The exogenous colony-forming units (CFUs) were significantly enhanced in the copper-deficient mice. The decreased splenic lymphoid populations, decreased anti-ss-DNA titers, and increased exogenous CFUs in the copper-deficient mice appear to be due to an increase in erythropoiesis at the expense of lymphopoiesis.

Familial Sneddon's syndrome: clinical, hematologic, and radiographic findings in two brothers.

We present the clinical, hematologic, and radiographic findings in two brothers with Sneddon's syndrome (stroke and livedo reticularis) and antiphospholipid antibodies. Patient 1 had anticardiolipin antibody and patient 2 had lupus anticoagulant, which we detected only upon repeated blood testing. One should test for both anticardiolipin antibody and lupus anticoagulant and repeat the screenings before determining a Sneddon's syndrome patient's antiphospholipid antibody status. Both Sneddon's syndrome and the primary antiphospholipid antibody syndrome are potentially familial causes of stroke. In familial cases, an inherited predisposition to antiphospholipid antibody production may be involved in disease pathogenesis.

The utility of spirometry in the diagnosis of reversible airways obstruction.

Patients with suspected reversible airways obstruction (RAO) sometimes report subjective benefit after bronchodilator treatment despite no objective spirometric improvement. One possible explanation for this is improvement in volume-related or plethysmographic parameters in the absence of spirometric improvement. One hundred patients with RAO were assessed before and after inhaled bronchodilator to determine the prevalence of improvement by plethysmographic parameters in the absence of improvement in spirometric parameters. Spirometry alone (FEV1, FVC, and FEF25-75%) identified reversibility of airflow limitation in 82 patients. Reversibility was identified by body plethysmography (specific conductance [SGaw], thoracic gas volume [TGV], and isovolume maximum expiratory flow rates [IVMEF]) in 15 of the remaining patients. The percent predicted FEF25-75% at baseline was higher in patients who required plethysmography to identify reversibility, but could not be used to predict the lack of a spirometric response for any individual patient. We conclude that spirometry alone fails to identify reversibility in approximately 15 percent of patients, and that most of these patients can be identified by additional plethysmographic measurements of volume-related parameters. At any one point in time, multiple tests must be used together to adequately identify the majority of patients with reversible airways obstruction. Improvement in volume-related parameters may explain why some patients with RAO improve subjectively with bronchodilators but show no spirometric improvement.

Evaluation of a Salmonella-specific DNA probe by colony hybridization using non-isotopic and isotopic labeling.

A 2.3 kilobase (kb) Salmonella probe, JEO402-1, and two subfragments, F1214 (1.3 kb) and F1217 (0.8 kb), have been evaluated by colony hybridization using pure cultures of Salmonella serovars and non-salmonella bacteria. JEO402-1, and its subfragments, F1214 and F1217, hybridized to all of 156 different Salmonella serovars tested, while there was no reaction to 112 non-salmonella strains belonging to 19 genera and 37 species of Enterobacteriaceae. Together with previously published results, the JEO402-1 probe has now been shown to detect a total of 396 Salmonella strains belonging to 214 serovars of Salmonella subspecies I-VI. A total of 178 non-salmonella strains representing 23 genera and 51 species of Enterobacteriaceae have all tested negative with JEO402-1. The hybridization results obtained using a digoxigenin-labeled probe were similar to those obtained with 35S isotopic labeling when complete colony lysis was ensured.

Virulence properties of Enterobacteriaceae isolated from the small intestine of children with diarrhea.

Enterobacteriaceae isolated from the duodena of Peruvian children with persistent diarrhea (PD) have been examined for virulence factors and compared with Enterobacteriaceae isolated from children with acute diarrhea, those convalescent from PD and diarrhea-free controls. Escherichia coli were isolated from 42 of 186 (23%) of the aspirates. All 11 children with PD in whom multiple E. coli colonies were examined were colonized by a single serotype. DNA probes identified enterotoxigenic E. coli in 2 of 89 (2.2%) PD aspirates and 2 of 38 (5.3%) acute diarrhea aspirates and enteroaggregative E. coli in one PD and one control aspirate. Strains positive with the enteropathogenic E. coli adherence factor probe were identified from 2 of 89 (2.2%) patients with PD and 1 of 34 (2.9%) controls. A subset of 12 E. coli strains failed to show adhesion to human duodenal enterocytes although 5 of 9 showed sparse but polar attachment to ileal cells from a child with short bowel syndrome and PD. Three of 10 Enterobacteriaceae (two E. coli, one Klebsiella species) caused diarrhea in the reversible ileal tie adult rabbit model. Colonization with virulent Enterobactericeae did not explain the majority of episodes of PD. Examination of these duodenal bacteria in the rabbit model revealed some that caused diarrhea but were not recognized pathogens.

Antibodies to Escherichia coli O157 in patients with haemorrhagic colitis and haemolytic uraemic syndrome.

Twenty four sera from patients with haemolytic uraemic syndrome or haemorrhagic colitis and healthy controls were examined for antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157. Faecal specimens from these patients were also examined for Vero cytotoxin producing E coli (VTEC) by DNA probes, and for faecal Vero cytotoxin. Eight patients with faecal E coli O157:H7 gave a strong antibody response to O157 LPS, shown by immunoblotting and an enzyme linked immunosorbent assay. Six symptomatic patients without evidence of faecal VTEC also gave a strong antibody response to O157 LPS. Sera from the remaining five patients and five healthy controls did not contain antibodies to E coli O157. The results suggest that the testing of sera from patients with haemorrhagic colitis or haemolytic uraemic syndrome by ELISA or immunoblot would prove valuable in addition to the established procedures for detecting VTEC, using DNA probes and testing for faecal Vero cytotoxin.