Adnexal torsion recurrence-is the adnexal twist degree a risk factor? A retrospective cohort study.

OBJECTIVE: To evaluate whether the adnexal twist degree is related to torsion recurrence and whether there is a dose-dependent correlation. DESIGN: A retrospective cohort study. SETTING: Single tertiary medical centre. POPULATION: The study includes non-pregnant patients operated, for the first time, for adnexal torsion, between 2011 and 2018. METHODS: Information regarding the degree of adnexal twist was collected from surgical reports. Recurrence was identified using a computerised database and ascertained via telephone with a response rate of 87.2% (253/290). MAIN OUTCOME MEASURES: Adnexal torsion recurrence rate. RESULTS: A total of 182 women who had undergone laparoscopic detorsion met the inclusion criteria. Twenty-two had torsion recurrence (12.1%). Adnexal twist degree in the primary event was associated with a higher recurrence risk: 4.3% of women with twist degree ≤360 (n = 3/70), 14.5% of women with twist degree of 361-720 (n = 9/62) and 20% of women with twist degree >720 (n = 10/50) (P = 0.03). The median twist degree was 540 (interquartile range [IQR] 360-855) and 720 (IQR 675-1080) degrees in the control and study groups, respectively (P = 0.005). Additional possibly associated factors for recurrence were evaluated. Age emerged as a possible risk factor, with a median age of 19 years in the recurrence group (IQR 14-27 years) versus 28.5 (IQR 19-36 years) in the non-recurrence group (P < 0.01). Logistic regression analysis revealed that together with age, adnexal twist degree remained significantly associated with torsion recurrence (odds ratio [OR] 1.98, 95% CI 1.09-3.61; P = 0.02). CONCLUSION: Adnexal twist degree was found to be positively associated with the risk of torsion recurrence. TWEETABLE ABSTRACT: Adnexal twist degree was found to be positively associated with the risk of torsion recurrence.

A systematic review of colorectal multidisciplinary team meetings: an international comparison.

BACKGROUND: Colorectal multidisciplinary teams (CR MDTs) were introduced to enhance the cancer care pathway and allow for early investigation and treatment of cancer. However, there are no 'gold standards' set for this process. The aim of this study was to review the literature systematically and provide a qualitative analysis on the principles, organization, structure and output of CR MDTs internationally. METHODS: Literature on the role of CR MDTs published between January 1999 and March 2020 in the UK, USA and continental Europe was evaluated. Historical background, structure, core members, education, frequency, patient-selection criteria, quality assurance, clinical output and outcomes were extracted from data from the UK, USA and continental Europe. RESULTS: Forty-eight studies were identified that specifically met the inclusion criteria. The majority of hospitals held CR MDTs at least fortnightly in the UK and Europe by 2002 and 2005 respectively. In the USA, monthly MDTs became a mandatory element of cancer programmes by 2013. In the UK, USA and in several European countries, the lead of the MDT meeting is a surgeon and core members include the oncologist, specialist nurse, histopathologist, radiologist and gastroenterologist. There were differences observed in patient-selection criteria, in the use of information technology, MDT databases and quality assurance internationally. CONCLUSION: CR MDTs are essential in improving the patient care pathway and should express clear recommendations for each patient. However, a form of quality assurance should be implemented across all MDTs.

Pentoxifylline mitigates detrimental impact of chronic nonbacterial prostatitis on sperm characteristics, reproductive hormones and histopathology in rats.

The protective role of pentoxifylline (PTX) on sperm characteristics, reproductive hormones and histopathology following carrageenan-induced chronic nonbacterial prostatitis (CNP) was investigated in male Wistar rats. Thirty-six rats were grouped into six rats per group. Group 1 (control) received saline normal. Group 2 received a single intraprostatic dose of 3% carrageenan (50 µl) on day 1 (CNP). Groups 3 and 5 received cernilton (standard drug) and PTX orally at 100 and 50 mg/kg for 14 consecutive days respectively. Groups 4 and 6 received a single dose of 3% carrageenan (50 µl) intraprostatically on day 1 followed by cernilton and PTX orally at 100 and 50 mg/kg on the eighth day for 14 consecutive days respectively. Prostatic index, serum prostatic specific antigen, malondialdehyde, testosterone and luteinising hormone levels were significantly increased (p < .05), whereas serum follicle-stimulating hormone, sperm count, motility and viability were significantly decreased (p < .05) in CNP group. Histopathology of prostate revealed leucocyte infiltration, large involutions and projection into the lumen in CNP group and these aberrations were improved by PTX. According to these findings, we concluded that PTX effectively mitigated detrimental impact of CNP on sperm characteristics, reproductive hormones and histopathology in rats.

[Management of complications after revascularization due to peripheral arterial occlusive disease : Prophylaxis and consistent adequate therapy after timely diagnostics]. / Management von Komplikationen nach Revaskularisation wegen peripherer arterieller Verschlusskrankheit : Prophylaxe und konsequente adäquate Therapie nach zeitgerechter Diagnostik.

BACKGROUND: Complications are a threat to successful revascularization for treatment of perpheral arterial occlusive disease (PAOD) and must, therefore, be either primarily prevented or effectively treated after having occurred. OBJECTIVES: The aim of this article is to give a survey of possible complications after revascularization for treatment of PAOD and their management. MATERIAL AND METHODS: A systematic literature review was performed in PubMed and Medline. The analysis mainly considered recent publications with a higher level of evidence. RESULTS: Revascularization for treatment of PAOD can basically be performed by an open surgical approach, an endovascular approach or as a combination of both methods (hybrid operation). The spectrum of possible complications varies accordingly. A differentiation can be made between bleeding, ischemic and systemic complications as well as between vascular and non-vascular complications. Optimal management of complications begins with primary prophylaxis and further includes a timely diagnosis and treatment of established complications. The best prophylaxis consists of a high quality of indications and performance of revascularization. CONCLUSION: Optimal management of complications is essential and of utmost importance for successful revascularization to treat PAOD.

Active eosinophilic esophagitis is characterized by epithelial barrier defects and eosinophil extracellular trap formation.

BACKGROUND: Eosinophilic esophagitis (EoE) exhibits esophageal dysfunction owing to an eosinophil-predominant inflammation. Activated eosinophils generate eosinophil extracellular traps (EETs) able to kill bacteria. There is evidence of an impaired barrier function in EoE that might allow pathogens to invade the esophagus. This study aimed to investigate the presence and distribution of EETs in esophageal tissues from EoE patients and their association with possible epithelial barrier defects. METHODS: Anonymized tissue samples from 18 patients with active EoE were analyzed. The presence of DNA nets associated with eosinophil granule proteins forming EETs and the expression of filaggrin, the protease inhibitor lympho-epithelial Kazal-type-related inhibitor (LEKTI), antimicrobial peptides, and cytokines were evaluated by confocal microscopy following immune fluorescence staining techniques. RESULTS: Eosinophil extracellular trap formation occurred frequently and was detected in all EoE samples correlating with the numbers of infiltrating eosinophils. While the expression of both filaggrin and LEKTI was reduced, epithelial antimicrobial peptides (human beta-defensin-2, human beta-defensin-3, cathelicidin LL-37, psoriasin) and cytokines (TSLP, IL-25, IL-32, IL-33) were elevated in EoE as compared to normal esophageal tissues. There was a significant correlation between EET formation and TSLP expression (P = 0.02) as well as psoriasin expression (P = 0.016). On the other hand, a significant negative correlation was found between EET formation and LEKTI expression (P = 0.016). CONCLUSION: Active EoE exhibits the presence of EETs. Indications of epithelial barrier defects in association with epithelial cytokines are also present which may have contributed to the activation of eosinophils. The formation of EETs could serve as a firewall against the invasion of pathogens.

The generation of neutrophils in the bone marrow is controlled by autophagy.

Autophagy has been demonstrated to have an essential function in several cellular hematopoietic differentiation processes, for example, the differentiation of reticulocytes. To investigate the role of autophagy in neutrophil granulopoiesis, we studied neutrophils lacking autophagy-related (Atg) 5, a gene encoding a protein essential for autophagosome formation. Using Cre-recombinase mediated gene deletion, Atg5-deficient neutrophils showed no evidence of abnormalities in morphology, granule protein content, apoptosis regulation, migration, or effector functions. In such mice, however, we observed an increased proliferation rate in the neutrophil precursor cells of the bone marrow as well as an accelerated process of neutrophil differentiation, resulting in an accumulation of mature neutrophils in the bone marrow, blood, spleen, and lymph nodes. To directly study the role of autophagy in neutrophils, we employed an in vitro model of differentiating neutrophils that allowed modulating the levels of ATG5 expression, or, alternatively, intervening pharmacologically with autophagy-regulating drugs. We could show that autophagic activity correlated inversely with the rate of neutrophil differentiation. Moreover, pharmacological inhibition of p38 MAPK or mTORC1 induced autophagy in neutrophilic precursor cells and blocked their differentiation, suggesting that autophagy is negatively controlled by the p38 MAPK-mTORC1 signaling pathway. On the other hand, we obtained no evidence for an involvement of the PI3K-AKT or ERK1/2 signaling pathways in the regulation of neutrophil differentiation. Taken together, these findings show that, in contrast to erythropoiesis, autophagy is not essential for neutrophil granulopoiesis, having instead a negative impact on the generation of neutrophils. Thus, autophagy and differentiation exhibit a reciprocal regulation by the p38-mTORC1 axis.

[Aneurysms of the abdominal aorta and iliac arteries: paradigm shift - operative therapy, if possible endovascular?]. / Aneurysmen der Bauchaorta und der Beckenarterien: Paradigmenwechsel - operative Therapie, wenn möglich endovaskulär?

BACKGROUND: Therapy of abdominal aortic aneurysms (AAA) is currently based on a high level of evidence. This is not true in the same manner for iliac artery aneurysms (IAA) which are frequently associated with AAAs and occur only rarely as isolated lesions. The therapeutic principles apply in the same way to both aneurysm locations. OBJECTIVES: New findings, improved perioperative care and the rapid development of minimally invasive techniques require a constant update which is the aim of this article concerning the therapy of AAAs and IAAs. MATERIAL AND METHODS: A systematic literature review was performed in PubMed and Medline and priority was given to recent publications with a high level of evidence. RESULTS: Endovascular aneurysm repair (EVAR) and open aneurysm repair (OAR) result in a similar long-term survival. The perioperative survival advantage with EVAR persists only during medium-term postoperative courses. The reintervention rate after EVAR is substantially higher compared to OAR. For older patients and those who are considered unfit for OAR the expected benefits from EVAR has not been proven to date. Aneurysmal ruptures after EVAR demonstrate that a life-long surveillance of these patients is necessary. CONCLUSION: Therapy of AAAs and IAAs is increasingly being performed by EVAR. Even the majority of complex aneurysms are amenable to minimally invasive treatment. Nevertheless, indications for OAR continue to exist. Screening for AAAs results in a decrease of aneurysmal ruptures for which EVAR is also gaining importance.

p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene.

p73, a member of the p53 tumor suppressor family, is involved in neurogenesis, sensory pathways, immunity, inflammation, and tumorigenesis. How p73 is able to participate in such a broad spectrum of different biological processes is still largely unknown. Here, we report a novel role of p73 in regulating lipid metabolism by direct transactivation of the promoter of autophagy-related protein 5 (ATG5), a gene whose product is required for autophagosome formation. Following nutrient deprivation, the livers of p73-deficient mice demonstrate a massive accumulation of lipid droplets, together with a low level of autophagy, suggesting that triglyceride hydrolysis into fatty acids is blocked owing to deficient autophagy (macrolipophagy). Compared with wild-type mice, mice functionally deficient in all the p73 isoforms exhibit decreased ATG5 expression and lower levels of autophagy in multiple organs. We further show that the TAp73α is the critical p73 isoform responsible for inducing ATG5 expression in a p53-independent manner and demonstrate that ATG5 gene transfer can correct autophagy and macrolipophagy defects in p73-deficient hepatocytes. These data strongly suggest that the p73-ATG5 axis represents a novel, key pathway for regulating lipid metabolism through autophagy. The identification of p73 as a major regulator of autophagy suggests that it may have an important role in preventing or delaying disease and aging by maintaining a homeostatic control.

Thymic stromal lymphopoietin stimulates the formation of eosinophil extracellular traps.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) that is released by epithelial cells upon certain environmental triggers activates cells of the innate and adaptive immune system resulting in a preferential T helper 2 immune response. By releasing eosinophil extracellular traps (EETs), eosinophils achieve an efficient extracellular bacterial killing. Eosinophil extracellular traps release, however, has been observed in both infectious and noninfectious eosinophilic diseases. Here, we aim to investigate whether eosinophils generate functional EETs as a direct response to TSLP, and further to study the extra- and intracellular mechanisms involved in this process as well as TSLP receptor (TSLPR) expression by eosinophils in vitro and in vivo. METHODS: Thymic stromal lymphopoietin receptor expression on blood and tissue eosinophils was assessed by immunoblotting, flow cytometry, and immunofluorescence staining. Purified eosinophils were stimulated with recombinant human TSLP. The release of extracellular DNA in association with eosinophilic cationic protein (ECP) was detected by fluorescence staining techniques and confocal microscopy. In addition, cell survival, cell adhesion, production of reactive oxygen species (ROS), and the inhibition of bacterial growth by TSLP-stimulated eosinophils were measured. RESULTS: Thymic stromal lymphopoietin receptor was observed on peripheral blood eosinophils as well as on tissue infiltrating eosinophils in skin diseases. TSLP did not affect eosinophil survival, but induced the formation of EETs consisting of mitochondrial DNA in association with ECP in a concentration- and time-dependent manner. Eosinophil extracellular trap release could be inhibited by blocking either cell adhesion or ROS production. While eosinophils prevented the growth of both Staphylococcus aureus and Staphylococcus epidermidis, the latter were unable to elicit EET formation and eosinophils required additional TSLP stimulation to achieve this antibacterial activity. CONCLUSIONS: thymic stromal lymphopoietin directly stimulates eosinophils to produce EETs. Our observations link epithelial TSLP expression triggered by environmental factors with pathogen defense mechanisms involving eosinophils.

Thrombus in the non-aneurysmal, non-atherosclerotic descending thoracic aorta--an unusual source of arterial embolism.

INTRODUCTION: Mural thrombus of the thoracic aorta is a rare clinical finding in the absence of aneurysm or atherosclerosis. METHODS: The medical records of all patients diagnosed with a thrombus of a non-aneurysmatic and non-atherosclerotic descending thoracic aorta (NAADTA) and treated by the senior author between 04/1997 and 04/2010 were reviewed. RESULTS: Eight patients with mural thrombus of the NAADTA were identified. Arterial embolism was the main clinical finding in all cases and involved the lower extremities (n = 6), mesenteric (n = 3) or renal arteries (n = 2). Hypercoagulable disorders were present in 3 cases and a concurrent malignancy in another 3. Two patients underwent open surgery while 4 patients were treated conservatively with anticoagulation. Of the remaining 2 patients, one was treated with a thoracic stent-graft and aorto-biiliac bypass and the other one with transfemoral thrombectomy. Technical success was achieved in all surgical cases and thrombus resolution or stable disease in the conservative management group. No thrombus recurrence was observed during a mean follow-up of 49 months. CONCLUSION: The management of mural thrombus in NAADTA represents a challenge, especially in case of malignant disease or hypercoagulable disorder as a potential underlying pathology and should be individualized. Although no consensus exists in the literature, therapeutic anticoagulation is proposed as first-line therapy. The indication for surgical intervention results from contraindication to anticoagulation, mobile thrombus or recurrent embolism. Whenever possible, endovascular therapy should be preferred.

[Role of endovascular therapy for redo surgery in patients after aortoiliac aneurysm exclusion]. / Stellenwert der endovaskulären Therapie bei Reeingriffen nach Ausschaltung aortoiliakaler Aneurysmata.

INTRODUCTION: Redo surgery or reintervention following conventional or endovascular aortoiliac reconstruction often requires exclusion of new aneurysms. In the present study the potentials of endovascular management of such lesions are investigated. METHODS: All patients with endovascular reoperation for of newly developed aortoiliac aneurysms were identified from a prospectively run data-base. The indications and results of endovascular therapy were analysed retrospectively. In detail, data were analysed for the type of original operation, interval until and kind of reoperation, and results concerning survival, technical success and complications. RESULTS: From 12 / 2003 through 3 / 2007 195 patients with aortoiliac aneurysms were operated. Endovascular repair was performed in 15 cases of previously excluded aneurysms. Mean age of these 15 patients (12 men) was 73 (64-85) years. Ten patients had a primary conventional (group A) and 5 patients had a primary endovascular (group B) aneurysm repair. The mean time interval between the first and second operation was 8.9 (1-26) years. The secondary endovascular therapy in group A was successful in all cases. In group B endoleaks type I a (n = 1), I a / b (n = 1), II (n = 2) and III (n = 1) were treated. One type II endoleak could only be treated successfully by conversion to open repair, the other one was successfully treated by reintervention. All but one patient are alive and -remained free of pathological findings during a median follow-up of 13 (2-39) months. DISCUSSION: Because of the clearly elevated operation risk of redo surgery after conventional or endovascular aneurysm repair, endovascular aneurysm exclusion represents the method of first choice. The reasonable selection and combination of procedures allows for an optimal adaptation of therapy to the individual case.

Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.

A novel FIP1L1-PDGFRA mutant destabilizing the inactive conformation of the kinase domain in chronic eosinophilic leukemia/hypereosinophilic syndrome.

BACKGROUND: The Fip1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRA) gene fusion is a common cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES), and patients suffering from this particular subgroup of CEL/HES respond to low-dose imatinib therapy. However, some patients may develop imatinib resistance because of an acquired T674I mutation, which is believed to prevent drug binding through steric hindrance. METHODS: In an imatinib resistant FIP1L1-PDGFRA positive patient, we analyzed the molecular structure of the fusion gene and analyzed the effect of several kinase inhibitors on FIP1L1-PDGFRA-mediated proliferative responses in vitro. RESULTS: Sequencing of the FIP1L1-PDGFRA fusion gene revealed the occurrence of a S601P mutation, which is located within the nucleotide binding loop. In agreement with the clinical observations, imatinib did not inhibit the proliferation of S601P mutant FIP1L1-PDGFRA-transduced Ba/F3 cells. Moreover, sorafenib, which has been described to inhibit T674I mutant FIP1L1-PDGFRA, failed to block S601P mutant FIP1L1-PDGFRA. Structural modeling revealed that the newly identified S601P mutated form of PDGFRA destabilizes the inactive conformation of the kinase domain that is necessary to bind imatinib as well as sorafenib. CONCLUSIONS: We identified a novel mutation in FIP1L1-PDGFRA resulting in both imatinib and sorafenib resistance. The identification of novel drug-resistant FIP1L1-PDGFRA variants may help to develop the next generation of target-directed compounds for CEL/HES and other leukemias.

Neutrophil apoptosis mediated by nicotinic acid receptors (GPR109A).

G protein-coupled receptor (GPR)109A (HM74A) is a G(i) protein-coupled receptor, which is activated by nicotinic acid (NA), a lipid-lowering drug. Here, we demonstrate that mature human neutrophils, but not eosinophils, express functional GPR109A receptors. The induction of the GPR109A gene appears to occur late in the terminal differentiation process of neutrophils, since a mixed population of immature bone marrow neutrophils did not demonstrate evidence for its expression. NA accelerated apoptosis in cultured neutrophils in a concentration-dependent manner, as assessed by phosphatidylserine redistribution, caspase-3 activation, and DNA fragmentation assays. The pro-apoptotic effect of NA was abolished by pertussis toxin, which was used to block G(i) proteins, suggesting a receptor-mediated mechanism. Activation of GPR109A by NA resulted in decreased levels of cyclic adenosine monophosphate (cAMP), most likely due to G(i)-mediated inhibition of adenylyl cyclase activity. NA-induced apoptosis was reversed by the addition of cell-permeable cAMP, pointing to the possibility that reduced cAMP levels promote apoptosis in neutrophils. Distal mechanism involved in this process may include the post-translational modification of members of the Bcl-2 family, such as dephosphorylation of pro-apoptotic Bad and antiapoptotic Mcl-1 proteins. Taken together, following maturation in the bone marrow, neutrophils express functional GPR109A receptors, which might be involved in the regulation of neutrophil numbers. Moreover, this study identified a new cellular target of NA and future drugs activating GPR109A receptors, the mature neutrophil.

Targeting survivin via PI3K but not c-akt/PKB by anticancer drugs in immature neutrophils.

Myelosuppression is the most common unwanted side effect associated with the administration of anticancer drugs, and infections remain a common cause of death in chemotherapy-treated patients. Several mechanisms of the cytotoxicity of these drugs have been proposed and may synergistically operate in a given cell. Survivin expression has been associated with cancer, but recent reports suggest that this molecule is also expressed in several immature and mature hematopoietic cells. Here, we provide evidence that treatment of immature neutrophils with anticancer drugs reduced endogenous survivin levels causing apoptosis. The anticancer drugs did not directly target survivin, instead they blocked the activity of phosphatidylinositol-3-OH kinase, which regulated survivin expression and apoptosis in these cells. Strikingly, and in contrast to other cells, this pathway did not involve the serine/threonine kinase c-akt/PKB. Moreover, in combination with anticancer drug therapy, rapamycin did not induce increased myelosuppression in an experimental lymphoma mouse model. These data suggest that drugs that block either c-akt/PKB or signaling molecules located distal to c-akt/PKB may preferentially induce apoptosis of cancer cells as they exhibit no cytotoxicity for immature neutrophils.

Citrate shows specific, dose-dependent lympholytic activity in neoplastic cell lines.

We have demonstrated cell membrane destruction activity by carboxylic acid derivatives (CADs) mainly tri-sodium citrate, in neoplastic cell lines and, to a far lesser extent, in normal human peripheral blood mononuclear cells (hPBMC). Flow cytometric (FACS) analysis was applied to Annexin-V and Propidium Iodide (PI) stained cells to evaluate the degree of the apoptosis induced by citrate in the following cell lines: CCRF-CEM (shortened to CEM), H9, and Jurkat (T-Cells), Raji and WIL2-NS (B-Cells), HL-60 (myeloblasts), K562 (myelocytes) and U937 (monocytes). We also tested normal hPBMC. Before staining with Annexin/PI, manual cell counts were performed on 24- and 48-h-old cell cultures. Cell supernatants were assayed for lactate dehydrogenase (LDH). LDH values in samples correlated with enhanced apoptosis by FACS analysis. For comparison, ascorbate and 2 other CADs including, acetate and lactate were also evaluated for the induction of apoptosis. In addition, the ability of tri-sodium citrate to induce apoptosis in the presence and the absence of several antineoplastic drugs, such as dexamethasone, arsenic trioxide, hydrocortisone, 6-mercaptopurine, and methotrexate were tested on Jurkat cells. FACS, LDH, and cell count values all demonstrated an enhanced degree of apoptotic cell death in Jurkat cells by citrate. In most of our investigated cells, except for the H9 cell line, citrate has induced a greater degree of apoptosis than acetate which induced a greater degree than lactate (see Fig. 1.0). The nature of the cell death by ascorbate appeared to be due to necrosis rather than apoptosis. Pilot studies on normal hPBMC showed that citrate alone or in combination with antineoplastic drugs caused minimal cell death. Thus citrate might be of benefit in some chemotherapy treatments in order to reduce drug toxicity or possibly enhance drug activities in certain neoplasias.

HIV-1 infection is facilitated in T cells by decreasing p56lck protein tyrosine kinase activity.

Several studies have suggested an important role for the protein tyrosine kinase p56lck (Lck) in HIV infection; however, the exact nature of this role remains unclear. Using a series of well characterized Jurkat-derived cell lines having a wide range of Lck kinase activity, our results showed that, while the entry of HIV-1 into these cell lines was similar, the kinetics of virus production by these cells were very different. Cells expressing a kinase-inactive Lck showed accelerated viral replication, whereas, cells expressing Lck with normal or elevated enzymatic activity showed a delay in virus replication that was proportional to the initial level of endogenous Lck activity. The cell line having the highest initial Lck kinase activity showed the slowest rate of productive HIV-1 infection. Analysis of 2-LTR circles revealed that this inhibitory effect of Lck was not due to inhibition of reverse transcription of HIV-1 genome or migration of the proviral DNA into the nuclei. This affect of Lck was confirmed in additional studies that used either the S1T cell line lacking completely Lck or where the Lck activity was altered in Jurkat cells prior to infection. S1T cells showed a 3- to 12-fold increase in the level of infection compared to Jurkat cells despite similar CD4 and chemokine coreceptor expression and cell doubling times. Pretreatment of Jurkat with an antisense lck oligodeoxynucleotide inhibited the synthesis of functional Lck and facilitated the viral replication by the cells as did expressing a dominant-negative mutant Lck which increased the productive infection>3-fold. Conversely, whereas IL-16 had no affect on productive infection in S1T cells that lack Lck, IL-16 pretreatment of Jurkat cells resulted in an immediate (within 5 min) and sustained and gradual (over 5 h) increase in Lck activity that resulted in a reduction of HIV-1 replication that paralleled the increasing Lck kinase activity. These results show that the enzymatic activity of Lck kinase can affect viral replication, that a lack of, or decreased Lck activity facilitates viral replication. Conversely, Lck can mediate a delay in HIV-1 infection that is proportional to the initial endogenous Lck enzyme activity.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.