Repurposing BCL-2 and Jak 1/2 inhibitors: Cure and treatment of HIV-1 and other viral infections.

B cell lymphoma 2 (BCL-2) family proteins are involved in the mitochondrial apoptotic pathway and are key modulators of cellular lifespan, which is dysregulated during human immunodeficiency virus type 1 (HIV-1) and other viral infections, thereby increasing the lifespan of cells harboring virus, including the latent HIV-1 reservoir. Long-lived cells harboring integrated HIV-1 DNA is a major barrier to eradication. Strategies reducing the lifespan of reservoir cells could significantly impact the field of cure research, while also providing insight into immunomodulatory strategies that can crosstalk to other viral infections. Venetoclax is a first-in-class orally bioavailable BCL-2 homology 3 (BH3) mimetic that recently received Food and Drug Administration (FDA) approval for treatment in myeloid and lymphocytic leukemia. Venetoclax has been recently investigated in HIV-1 and demonstrated anti-HIV-1 effects including a reduction in reservoir size. Another immunomodulatory strategy towards reduction in the lifespan of the reservoir is Jak 1/2 inhibition. The Jak STAT pathway has been implicated in BCL-2 and interleukin 10 (IL-10) expression, leading to a downstream effect of cellular senescence. Ruxolitinib and baricitinib are FDA-approved, orally bioavailable Jak 1/2 inhibitors that have been shown to indirectly decay the HIV-1 latent reservoir, and down-regulate markers of HIV-1 persistence, immune dysregulation and reservoir lifespan in vitro and ex vivo. Ruxolitinib recently demonstrated a significant decrease in BCL-2 expression in a human study of virally suppressed people living with HIV (PWH), and baricitinib recently received emergency use approval for the indication of coronavirus disease 2019 (COVID-19), underscoring their safety and efficacy in the viral infection setting. BCL-2 and Jak 1/2 inhibitors could be repurposed as immunomodulators for not only HIV-1 and COVID-19, but other viruses that upregulate BCL-2 anti-apoptotic proteins. This review examines potential routes for BCL-2 and Jak 1/2 inhibitors as immunomodulators for treatment and cure of HIV-1 and other viral infections.

Targeting Macrophage Dysregulation for Viral Infections: Novel Targets for Immunomodulators.

A major barrier to human immunodeficiency virus (HIV-1) cure is the latent viral reservoir, which persists despite antiretroviral therapy (ART), including across the non-dividing myeloid reservoir which is found systemically in sanctuary sites across tissues and the central nervous system (CNS). Unlike activated CD4+ T cells that undergo rapid cell death during initial infection (due to rapid viral replication kinetics), viral replication kinetics are delayed in non-dividing myeloid cells, resulting in long-lived survival of infected macrophages and macrophage-like cells. Simultaneously, persistent inflammation in macrophages confers immune dysregulation that is a key driver of co-morbidities including cardiovascular disease (CVD) and neurological deficits in people living with HIV-1 (PLWH). Macrophage activation and dysregulation is also a key driver of disease progression across other viral infections including SARS-CoV-2, influenza, and chikungunya viruses, underscoring the interplay between macrophages and disease progression, pathogenesis, and comorbidity in the viral infection setting. This review discusses the role of macrophages in persistence and pathogenesis of HIV-1 and related comorbidities, SARS-CoV-2 and other viruses. A special focus is given to novel immunomodulatory targets for key events driving myeloid cell dysregulation and reservoir maintenance across a diverse array of viral infections.

Ischaemic breast necrosis following coronary artery bypass grafting using left internal mammary artery: understanding the risks.

BACKGROUND: Coronary artery bypass grafting (CABG) is a common procedure performed commonly using left internal mammary artery (LIMA). We report a case of sternal wound dehiscence and breast necrosis following LIMA harvest in a 55-year-old obese lady with macromastia, diabetes mellitus, hypertension and end stage renal disease requiring dialysis. We also review the existing literature. METHODS: Publications were identified from Medline All, Web of Science Core Collection, Google Scholar and Cochrane Central Register of Controlled Trial between 1974 and 2 January 2020. We assessed patient co-morbidities, presentation time frame, quadrant of breast necrosis and reconstruction option utilized. RESULTS: There were 18 cases of breast necrosis reported post-CABG. The patients were aged 50 or over, morbidly obese and had large tubular breasts. Other risk factors included chronic renal insufficiency, diabetes and hypertension. The presentation was delayed with necrosis only evident 7 days or more after CABG. All the reported cases had necrosis at lower inner quadrant of the left breast with nipple-areola complex. CONCLUSION: Blood supply to the breast is segmental with considerable overlap, however, this overlap is reduced in large breasts. Patients with macromastia and multiple co-morbidities need preoperative imaging (computed tomography angiogram) to evaluate breast vascularity prior to CABG. Risk of breast necrosis needs to be explained during the informed consent. Surgical management of the hypermastia (breast reduction or amputation) may be a factor in facilitating the CABG procedure if indeed the LIMA is absolutely indicated considering the risks and benefits.

Burns infection profile of Singapore: prevalence of multidrug-resistant Acinetobacter baumannii and the role of blood cultures.

BACKGROUND: With various changes implemented such as perioperative antibiotics for tangential excision, this retrospective study reviews the infection profile of burn patients at Singapore's only centralized burns unit. Worldwide, the appearance of multidrug-resistant (MDR) strains of Acinetobacter baumannii (A. baumannii) continues to worsen patient outcomes. This study also surveys the role of blood cultures in burns at our unit. METHODS: Four hundred fifty-two burn patients admitted to the unit between 2011 and 2013, and with cultures performed, were included in the study. The yields of various cultures were evaluated and 2684 samples were amassed, of which 984 (36.7 %) were positive. Patient variables for predictors of MDR A. baumannii infection acquisition and bacteremia were evaluated through multivariate analyses. RESULTS: Pseuodomonas aeruginosa (P. aeruginosa) (67 patients) was the most common organism in those with total body surface area (TBSA) burn <20 % while MDR A. baumannii (39 patients) was most prevalent in those with TBSA burn ≥20 %. We found a yield of 1.1 % positive blood cultures for TBSA burn <20 % and a yield of 18.6 % positive cultures in TBSA burn ≥20 %. The median time between surgery and bacteremia was 6.5 days (range -18 to 68 days, interquartile range 4.5); 2.9 and 8.8 % of bacteremic episodes occurred within 24 and 48 h, respectively. This is a decrease from a predeceasing study (45.3 % for 24 h and 60 % for 48 h). Multivariate analysis revealed that length of hospital stay and TBSA burn ≥20 % were predictors of MDR A. baumannii infection and positive blood cultures. CONCLUSIONS: MDR A. baumannii infection burdens patient management, especially in those with TBSA burn ≥20 % and longer hospital stay. Prophylactic antibiotics may reduce perioperative bacteremia, but their role in MDR infections needs to be evaluated. The role of blood cultures in TBSA burn <20 % needs reconsideration.

Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

INTRODUCTION: There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. METHODS: We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. RESULTS: Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, ß1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. CONCLUSIONS: These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

Systematic review of seat-belt trauma to the female breast: a new diagnosis and management classification.

BACKGROUND: Legislation, education and technology have led to a rise in the use of seat belts. This has significantly reduced automobile accident-related mortality, but it has increased seat belt-associated injuries. The aim of this study was to review all publications on seat-belt injury to the female breast and to analyse patterns of presentation, management and outcomes. METHODS: A literature search was performed by two independent reviewers using the PubMed, Scopus and EMBASE databases. The MeSH terms included 'seat belt breast' or 'breast traffic accident' or 'safety belt breast'. This study was supported by Level V evidence. RESULTS: In this review, 26 articles describing 42 patients were included. A total of 13 patients (31.0%) presented immediately after the road traffic accident (RTA) with pain, swelling, open wounds and/or haemorrhage of the breast. Active arterial extravasations were treated with angiographic embolization while wounds were mostly treated with dressings. Twenty-nine (69.0%) patients presented late, with a mean time of 6.77 months (3 weeks-5 years) after the RTA. This ranged from a palpable lump in the area of trauma to a worsening cleft deformity. Most late presentations were diagnosed with fat necrosis while five patients (17.2%) were found to have breast cancer. From this review, we propose a four-tier classification system that categorizes patients based on timing to presentation and symptoms, with recommended investigation and management options for each category. CONCLUSION: This is the most comprehensive systemic review to date of seat-belt injuries to the female breast, and our proposed classification may be useful in the management of such patients.

Hypertrophic scars and keloids in surgery: current concepts.

Hypertrophic scars and keloids remain a challenge in surgery. We appreciate that our understanding of the process at cellular and molecular level, profound as it is, when it comes to the clinical evidence much is left to be desired. Although the bench to bedside conundrum remains, the science of translational research calls for an even higher level of cooperation between the scientist and the clinician for the impetus to succeed.The clinicians alerted us to the possible theories in the pathogenesis of keloid formation, inter alia, the ischemia theory, mast cell theory, immune theory, transforming growth factor ß interaction, mechanical theory, and the melanocyte stimulating hormone theory. All of the above presupposed a stimulus that would result in an uncontrolled upregulation of collagen and extracellular matrix expression in the pathogenesis of the keloid. This bedside to bench initiative, as in true science, realized more ponderables than possibilities.By the same token, research into the epidermal-mesenchymal signaling, molecular biology, genomics, and stem cell research holds much promise in the bench top arena. To assess efficacy, many scar assessment scores exist in the literature. The clinical measurement of scar maturity can aid in determining end points for therapeutics. Tissue oxygen tension and color assessment of scars by standardized photography proved to be useful.In surgery, the use of dermal substitutes holds some promise as we surmise that quality scars that arise from dermal elements, molecular and enzyme behavior, and balance. Although a systematic review shows some benefit for earlier closure and healing of wounds, no such review exists at this point in time for the use of dermal substitutes in scars.Adipose-derived stem cell, as it pertains to scars, will hopefully realize the potential of skin regeneration rather than by repair in which we are familiar with as well as the undesirable scarring as a result of healing through the inflammatory response.Translational research will bear the fruit of coordinating bench to bedside and vice versa in the interest of progress into the field of regenerative healing that will benefit the patient who otherwise suffers the myriad of scar complications.

Characterization of early thermal burns and the effects of hyperbaric oxygen treatment: a pilot study.

BACKGROUND AND AIMS: Studies investigating hyperbaric oxygen treatment (HBOT) to improve outcome in burns have been inconclusive. In this study, we aimed to characterize early thermal burns injury in adult patients with < 40% total body surface area (TBSA) and to determine the effects of HBOT administered within 24 h to 48 h of a burn injury. METHODS: Seventeen subjects were randomized into control (n = 9) and HBOT treatment (n = 8) arms. Burn depth, measured by laser Doppler imaging (LDI) and histologically, white blood cell (WBC) count and plasma cytokine inflammatory markers were assessed at 24 h (pre HBOT) and 48 h (post HBOT) post burn, as were immunohistochemistry and microbiology of burns tissue samples at 48 h post burn. RESULTS: WBC count and serum interleukin (IL)-1ß, IL-4, IL-6, IL-10 and interferon-γ were significantly elevated 24 h after burn, but no significant changes in any of these parameters were found with HBOT. HBOT had no significant effect on burn depth. Two HBOT patients and four control patients developed positive bacterial cultures. CONCLUSIONS: Slower than anticipated recruitment resulted in considerably fewer patients than planned being studied. Inflammatory markers were significantly increased at 24 h in patients with < 40% TBSA burn. Early HBOT had no apparent effects on any of the parameters measured in this small pilot study. HBOT may possibly have a broad-spectrum antimicrobial effect worthy of further study. We report our methodology in detail as a possible model for future burns studies.

Decellularized porcine saphenous artery for small-diameter tissue-engineered conduit graft.

Decellularized xenografts have been identified as potential scaffolds for small-diameter vascular substitutes. This study aimed to develop and investigate a biomechanically functional and biocompatible acellular conduit using decellularized porcine saphenous arteries (DPSAs), through a modified decellularization process using Triton X-100/NH4 OH solution and serum-containing medium. Histological and biochemical analysis indicated a high degree of cellular removal and preservation of the extracellular matrix. Bursting pressure tests showed that the DPSAs could withstand a pressure of 1854 ± 164 mm Hg. Assessment of in vitro cell adhesion and biocompatibility showed that porcine pulmonary artery endothelial cells were able to adhere and proliferate on DPSAs in static and rotational culture. After interposition into rabbit carotid arteries in vivo, DPSAs showed patency rates of 60% at 1 month and 50% at 3 months. No aneurysm and intimal hyperplasia were observed in any DPSAs. All patent grafts showed regeneration of vascular elements, and thrombotic occlusion was found to be the main cause of graft failure, probably due to remaining xenoantigens. In conclusion, this study showed the development and evaluation of a decellularization process with the potential to be used as small-diameter grafts.

Homograft banking in Singapore: two years of cardiovascular tissue banking in Southeast Asia.

Established in 2008, the National Cardiovascular Homograft Bank (NCHB) has been instrumental in creating an available supply of cardiovascular tissues for implantation in Singapore. This article introduces its collaboration with Singapore General Hospital Skin Bank Unit. The procedure of homograft recovery, processing, cryopreservation and quality assurance are presented. Since its establishment, the NCHB has followed the guidelines set by the Ministry of Health Singapore and the American Association of Tissue Banks. A total of 57 homografts had been recovered and 40 homografts were determined to be suitable for clinical use. The most significant reasons for non-clinical use are positive microbiological culture or unsuitable graft condition. Crucial findings prompted reviews and implementation of new procedures to improve the safety of homograft recipients. These include (1) a change in antibiotic decontamination regime from penicillin and streptomycin to amikacin and vancomycin after a review and (2) mandating histopathogical examination since the discovery of cardiac sarcoidosis in a previously undiagnosed donor. Further, the NCHB also routinely performs dengue virus screening, for donors suspected of dengue infection. Cultural factors which affect the donation rate are also briefly explored. By 2010, 31 homografts had been implanted into recipients with congenital or acquired heart valve conditions. More than half of these recipients were children. Post-operative outcomes had been encouraging, with no report of adverse events attributed to implanted homografts.

Human tissues contain CD141hi cross-presenting dendritic cells with functional homology to mouse CD103+ nonlymphoid dendritic cells.

Dendritic cell (DC)-mediated cross-presentation of exogenous antigens acquired in the periphery is critical for the initiation of CD8(+) T cell responses. Several DC subsets are described in human tissues but migratory cross-presenting DCs have not been isolated, despite their potential importance in immunity to pathogens, vaccines, and tumors and tolerance to self. Here, we identified a CD141(hi) DC present in human interstitial dermis, liver, and lung that was distinct from the majority of CD1c(+) and CD14(+) tissue DCs and superior at cross-presenting soluble antigens. Cutaneous CD141(hi) DCs were closely related to blood CD141(+) DCs, and migratory counterparts were found among skin-draining lymph node DCs. Comparative transcriptomic analysis with mouse showed tissue DC subsets to be conserved between species and permitted close alignment of human and mouse DC subsets. These studies inform the rational design of targeted immunotherapies and facilitate translation of mouse functional DC biology to the human setting.

Keloid fibroblasts are more sensitive to Wnt3a treatment in terms of elevated cellular growth and fibronectin expression.

BACKGROUND: Current evidence suggests the potential role of Wnt signalling in keloids pathogenesis but such literature remains scanty. We hypothesize that Wnt signalling is upregulated in keloid fibroblasts (KFs) and this promotes cellular growth, migration and extracellular matrix (ECM) production in such fibroblasts. OBJECTIVES: To verify the downregulation of secreted frizzled-related protein 1 (SFRP1), a Wnt inhibitor and test KFs sensitivity to Wnt3a treatment compared to NFs in terms of activation of Wnt/ß-catenin, cellular growth, migration and ECM expressions. Next, to investigate if ectopic expression of SFRP1 and treatment of quercetin in KFs can reverse their phenotypes. METHODS: Quantitative Real-time PCR and western blotting were used to verify SFRP1 expression in NFs and KFs. The fibroblasts were tested with Wnt3a conditioned media and its effects were tested for (1) the cells' sensitivity to direct Wnt signalling via the activation of TCF reporter assay and protein expression of ß-catenin, (2) cellular growth, (3) cell migration and (4) expressions of ECM components. Finally KFs were stably transduced with SFRP1 and treated with 2 doses of quercetin. RESULTS: Lower levels of SFRP1 were confirmed at mRNA and protein levels in KFs which partly explained their sensitivity to Wnt3a treatment in terms of higher Wnt activation, cellular growth and fibronectin expression. Interestingly, Wnt3a did not promote higher cell migration rate and increase in collagen I expression. Ectopic expression of SFRP1 and quercetin treatment was able to mitigate Wnt3a-mediated phenotype of KFs. CONCLUSIONS: Using SFRP1 or inhibitors of Wnt signalling might be one of the therapeutic solutions to treat keloid scarring.

The role of R-spondin2 in keratinocyte proliferation and epidermal thickening in keloid scarring.

Keloids are found only in humans and the underlying biochemical mechanisms of their pathogenesis remain unknown. R-spondins (Rspos) are a relatively new group of secreted proteins known to be Wnt/ß-catenin signaling agonists, but their role in keloids has yet to be elucidated. We investigated the expression levels of R-spondin2 (Rspo2) in cell lysates and conditioned media of monocultures and co-cultures of fibroblasts and keratinocytes derived from keloids and normal skin. In this study we found increased protein expression and secretion of Rspo2 in respective monocultures of keloid fibroblasts and keratinocytes when compared with their normal counterparts. Double-chamber co-culture experiments implicated the role of keloid keratinocytes (KKs) in the induction of Rspo2 secretion from fibroblasts because of epithelial-mesenchymal interactions. Addition of recombinant human Rspo2 in culture increased the proliferation of keratinocytes and it acted synergistically with Wnt3a through the canonical Wnt/ß-catenin pathway. Overexpression of Rspo2 in normal fibroblasts brought about thicker epidermis when compared with control fibroblasts in a skin organotypic culture model. This observation coincides with the hyperproliferative phenotype of thickened epidermis seen in keloids. Taken together, the results suggest the possible double paracrine action of KKs in inducing higher expression of Rspo2 in fibroblasts that promotes keratinocyte proliferation and epidermal thickening.

Modifications of the radial forearm flap phalloplasty for female-to-male gender reassignment.

The radial forearm flap remains the preferred technique for phalloplasty. From 1999 to 2009, 19 patients with primary female transsexualism underwent gender reassignment surgery at our center. The radial forearm flap phalloplasty is modified as a two-stage procedure, with prelamination of the neourethra on the donor forearm before microsurgical transfer 3 months later. At 5-year follow-up, patients were asked to complete a survey on the functional, aesthetic, and psychological results postsurgery. The radial forearm flap reliably provided sufficient bulk with stiffness for the neophallus with acceptable aesthetic appearance. We further describe technical modifications to reduce the rate of urethral strictures and fistulas. None of the patients regretted undergoing gender transformation. Patients are satisfied with the surgical result and generally prepared to accept its potential costs, in view of the significant psychological and legal benefits.

Use of the microdebrider for treatment of fibrous gynaecomastia.

BACKGROUND: In the quest for reduced scars and better aesthetic outcomes in minimally invasive surgical techniques for gynaecomastia, suction-assisted lipoplasty and ultrasound-assisted lipoplasty are now considered accepted recent advancements. Nevertheless, the fibrous glandular breast disc encountered in young, thin patients requires a separate peri-areolar incision as the disc cannot be removed with suction lipoplasty. The use of a microdebrider (powered shaving rotary device) is a potential solution to this problem. We present a series of eight patients with fibrous gynaecomastia that was successfully treated in this way. METHOD: The surgery is performed under general anaesthesia. The microdebrider cannula is used to remove the fibrous glandular breast tissue. Drains are inserted and fibrin glue is sprayed subcutaneously. Patients are discharged on the next day. Drains are removed on the 5th postoperative day. A compressive vest is worn for 6 weeks. (A video of the procedure can be seen on http://www.microflap.com/video3.asp). RESULTS: The eight patients were successfully treated. No bleeding, haematoma or seroma was encountered. All patients were satisfied with the results of the surgery. CONCLUSION: The microdebrider is a viable solution in the treatment of gynaecomastia with a fibrous breast disc. Excellent aesthetic results can be achieved with a single 3-mm incision.

The fibula osteoseptocutaneous flap incorporating the hemisoleus muscle for complex head and neck defects: anatomical study and clinical applications.

BACKGROUND: In patients with extensive bone and soft-tissue defects, the inclusion of the hemisoleus muscle with the fibula osteoseptocutaneous flap would provide the needed soft-tissue volume to the flap. This study evaluates the reliability and technical considerations for the inclusion of the hemisoleus with the fibula and skin paddle as a chimeric, peroneal artery-based flap. METHODS: The location and size of major arterial branches of the peroneal artery supplying the lateral hemisoleus muscle were investigated in 10 cadaveric injected lower limb specimens. The utility of this design was demonstrated in five clinical cases. RESULTS: The lateral hemisoleus was noted to be consistently supplied by large muscle branches from the peroneal artery, soleus vessels 1 (proximal) and 2 (distal). The mean diameter and distance from the origin of the peroneal artery for soleus vessels 1 and 2 were 1.8 mm and 2.1 cm, and 1.6 mm and 6.3 cm, respectively. The fibula osteoseptocutaneous flap incorporating the hemisoleus muscle was performed in five clinical cases. All were successful. Either soleus vessel 1 or soleus vessel 2 can be used as the pedicle to the muscle, depending on the specific reconstructive requirements for the reach and placement of the hemisoleus. CONCLUSIONS: The fibula osteoseptocutaneous flap incorporating the hemisoleus muscle can reliably be raised by preserving constant muscle branches that arise from the peroneal artery to supply the lateral hemisoleus. This flap provides the additional bulk in selected cases, with little additional donor-site morbidity.

The use of Urgotul in the treatment of partial thickness burns and split-thickness skin graft donor sites: a prospective control study.

The use of paraffin-impregnated gauze for burns and skin graft donor sites is commonly associated with wound adherence with consequent pain and trauma upon removal. This prospective clinical study was performed to evaluate a new class of lipido-colloid dressings (Urgotul) in promoting healing and in reducing tissue adherence. In a 6-month period, 25 consecutive patients were recruited. Two separate burn or donor sites on each patient were dressed with tulle-gras (TG) or Urgotul and covered with standard secondary dressings. Objective assessment of wounds by two reviewers, and patients' subjective assessments were recorded. Twenty-three (92%) patients were followed up for a mean of 3 months. Mean time to complete epithelialisation was 9.6 and 11.9 days for the Urgotul and TG sites respectively (P < 0.05). Bleeding was seen in 52% of Urgotul sites compared with 100% of the TG sites at first dressing change (P < 0.05). Patients reported 'moderate pain' during dressing change in 22% and 57% in the Urgotul and TG groups respectively (P < 0.05), with 35% of TG sites being 'very painful' requiring extra analgesia. We found that compared with TG, Urgotul was associated with faster epithelialisation, less pain and trauma (bleeding) during dressing changes.