Reduced expression of mitochondrial fumarate hydratase in progressive multiple sclerosis contributes to impaired in vitro mesenchymal stromal cell-mediated neuroprotection.

BACKGROUND: Cell-based therapies for multiple sclerosis (MS), including those employing autologous bone marrow-derived mesenchymal stromal cells (MSC) are being examined in clinical trials. However, recent studies have identified abnormalities in the MS bone marrow microenvironment. OBJECTIVE: We aimed to compare the secretome of MSC isolated from control subjects (C-MSC) and people with MS (MS-MSC) and explore the functional relevance of findings. METHODS: We employed high throughput proteomic analysis, enzyme-linked immunosorbent assays and immunoblotting, as well as in vitro assays of enzyme activity and neuroprotection. RESULTS: We demonstrated that, in progressive MS, the MSC secretome has lower levels of mitochondrial fumarate hydratase (mFH). Exogenous mFH restores the in vitro neuroprotective potential of MS-MSC. Furthermore, MS-MSC expresses reduced levels of fumarate hydratase (FH) with downstream reduction in expression of master regulators of oxidative stress. CONCLUSIONS: Our findings are further evidence of dysregulation of the bone marrow microenvironment in progressive MS with respect to anti-oxidative capacity and immunoregulatory potential. Given the clinical utility of the fumaric acid ester dimethyl fumarate in relapsing-remitting MS, our findings have potential implication for understanding MS pathophysiology and personalised therapeutic intervention.

Prolonged disorders of consciousness: a critical evaluation of the new UK guidelines.

In March 2020, the Royal College of Physicians in the UK published national guidelines on the management of patients with prolonged disorders of consciousness, updating their 2013 guidance 'particularly in relation to recent developments in assessment and management and … changes in the law governing … the withdrawal of clinically assisted nutrition and hydration'. The report's primary focus is on patients who could live for many years with treatment and care. This update, by a neurologist, an imaging neuroscientist, and a lawyer-ethicist, questions the document's rejection of any significant role for neuroimaging techniques including functional MRI and/or bedside EEG to detect covert consciousness in such patients. We find the reasons for this rejection unconvincing, given (i) the significant advances made in the use of this technology in recent years; and (ii) the wider scope for its use envisaged by the earlier (2018) guidelines issued by the American Academy of Neurology. We suggest that, since around one in five patients diagnosed with prolonged disorders of consciousness are in fact conscious enough to follow commands in a neuroimaging context (i.e. those who are 'covertly conscious' or those with 'cognitive motor dissociation'), and given the clinical, ethical and legal importance of determining whether patients with prolonged disorders of consciousness are legally competent or at least able to express their views and feelings, the guidance from the Royal College of Physicians requires urgent review.

The diagnosis of primary central nervous system vasculitis.

The diagnosis of primary central nervous system (CNS) vasculitis is often difficult. There are neither specific clinical features nor a classical clinical course, and no blood or imaging investigations that can confirm the diagnosis. Contrast catheter cerebral angiography is neither specific nor sensitive, yet still underpins the diagnosis in many published studies. Here we describe an approach to its diagnosis, emphasising the importance of obtaining tissue, and present for discussion a new, binary set of diagnostic criteria, dividing cases into only 'definite' primary CNS vasculitis, where tissue proof is available, and 'possible,' where it is not. We hope that these criteria will be modified and improved by discussion among experts, and that these (improved) criteria may then be adopted and used as the basis for future prospective studies of the clinical features and diagnosis of this difficult and dangerous disorder, particularly for coordinated multicentre therapeutic trials.

Bone marrow transplantation stimulates neural repair in Friedreich's ataxia mice.

OBJECTIVE: Friedreich's ataxia is an incurable inherited neurological disease caused by frataxin deficiency. Here, we report the neuroreparative effects of myeloablative allogeneic bone marrow transplantation in a humanized murine model of the disease. METHODS: Mice received a transplant of fluorescently tagged sex-mismatched bone marrow cells expressing wild-type frataxin and were assessed at monthly intervals using a range of behavioral motor performance tests. At 6 months post-transplant, mice were euthanized for protein and histological analysis. In an attempt to augment numbers of bone marrow-derived cells integrating within the nervous system and improve therapeutic efficacy, a subgroup of transplanted mice also received monthly subcutaneous infusions of the cytokines granulocyte-colony stimulating factor and stem cell factor. RESULTS: Transplantation caused improvements in several indicators of motor coordination and locomotor activity. Elevations in frataxin levels and antioxidant defenses were detected. Abrogation of disease pathology throughout the nervous system was apparent, together with extensive integration of bone marrow-derived cells in areas of nervous tissue injury that contributed genetic material to mature neurons, satellite-like cells, and myelinating Schwann cells by processes including cell fusion. Elevations in circulating bone marrow-derived cell numbers were detected after cytokine administration and were associated with increased frequencies of Purkinje cell fusion and bone marrow-derived dorsal root ganglion satellite-like cells. Further improvements in motor coordination and activity were evident. INTERPRETATION: Our data provide proof of concept of gene replacement therapy, via allogeneic bone marrow transplantation, that reverses neurological features of Friedreich's ataxia with the potential for rapid clinical translation. Ann Neurol 2018;83:779-793.

Association of Initial Disease-Modifying Therapy With Later Conversion to Secondary Progressive Multiple Sclerosis.

Importance: Within 2 decades of onset, 80% of untreated patients with relapsing-remitting multiple sclerosis (MS) convert to a phase of irreversible disability accrual termed secondary progressive MS. The association between disease-modifying treatments (DMTs), and this conversion has rarely been studied and never using a validated definition. Objective: To determine the association between the use, the type of, and the timing of DMTs with the risk of conversion to secondary progressive MS diagnosed with a validated definition. Design, Setting, and Participants: Cohort study with prospective data from 68 neurology centers in 21 countries examining patients with relapsing-remitting MS commencing DMTs (or clinical monitoring) between 1988-2012 with minimum 4 years' follow-up. Exposures: The use, type, and timing of the following DMTs: interferon beta, glatiramer acetate, fingolimod, natalizumab, or alemtuzumab. After propensity-score matching, 1555 patients were included (last follow-up, February 14, 2017). Main Outcome and Measure: Conversion to objectively defined secondary progressive MS. Results: Of the 1555 patients, 1123 were female (mean baseline age, 35 years [SD, 10]). Patients initially treated with glatiramer acetate or interferon beta had a lower hazard of conversion to secondary progressive MS than matched untreated patients (HR, 0.71; 95% CI, 0.61-0.81; P < .001; 5-year absolute risk, 12% [49 of 407] vs 27% [58 of 213]; median follow-up, 7.6 years [IQR, 5.8-9.6]), as did fingolimod (HR, 0.37; 95% CI, 0.22-0.62; P < .001; 5-year absolute risk, 7% [6 of 85] vs 32% [56 of 174]; median follow-up, 4.5 years [IQR, 4.3-5.1]); natalizumab (HR, 0.61; 95% CI, 0.43-0.86; P = .005; 5-year absolute risk, 19% [16 of 82] vs 38% [62 of 164]; median follow-up, 4.9 years [IQR, 4.4-5.8]); and alemtuzumab (HR, 0.52; 95% CI, 0.32-0.85; P = .009; 5-year absolute risk, 10% [4 of 44] vs 25% [23 of 92]; median follow-up, 7.4 years [IQR, 6.0-8.6]). Initial treatment with fingolimod, alemtuzumab, or natalizumab was associated with a lower risk of conversion than initial treatment with glatiramer acetate or interferon beta (HR, 0.66; 95% CI, 0.44-0.99; P = .046); 5-year absolute risk, 7% [16 of 235] vs 12% [46 of 380]; median follow-up, 5.8 years [IQR, 4.7-8.0]). The probability of conversion was lower when glatiramer acetate or interferon beta was started within 5 years of disease onset vs later (HR, 0.77; 95% CI, 0.61-0.98; P = .03; 5-year absolute risk, 3% [4 of 120] vs 6% [2 of 38]; median follow-up, 13.4 years [IQR, 11-18.1]). When glatiramer acetate or interferon beta were escalated to fingolimod, alemtuzumab, or natalizumab within 5 years vs later, the HR was 0.76 (95% CI, 0.66-0.88; P < .001; 5-year absolute risk, 8% [25 of 307] vs 14% [46 of 331], median follow-up, 5.3 years [IQR], 4.6-6.1). Conclusions and Relevance: Among patients with relapsing-remitting MS, initial treatment with fingolimod, alemtuzumab, or natalizumab was associated with a lower risk of conversion to secondary progressive MS vs initial treatment with glatiramer acetate or interferon beta. These findings, considered along with these therapies' risks, may help inform decisions about DMT selection.

Cytokine therapy-mediated neuroprotection in a Friedreich's ataxia mouse model.

OBJECTIVES: Friedreich's ataxia is a devastating neurological disease currently lacking any proven treatment. We studied the neuroprotective effects of the cytokines, granulocyte-colony stimulating factor (G-CSF) and stem cell factor (SCF) in a humanized murine model of Friedreich's ataxia. METHODS: Mice received monthly subcutaneous infusions of cytokines while also being assessed at monthly time points using an extensive range of behavioral motor performance tests. After 6 months of treatment, neurophysiological evaluation of both sensory and motor nerve conduction was performed. Subsequently, mice were sacrificed for messenger RNA, protein, and histological analysis of the dorsal root ganglia, spinal cord, and cerebellum. RESULTS: Cytokine administration resulted in significant reversal of biochemical, neuropathological, neurophysiological, and behavioural deficits associated with Friedreich's ataxia. Both G-CSF and SCF had pronounced effects on frataxin levels (the primary molecular defect in the pathogenesis of the disease) and a regulators of frataxin expression. Sustained improvements in motor coordination and locomotor activity were observed, even after onset of neurological symptoms. Treatment also restored the duration of sensory nerve compound potentials. Improvements in peripheral nerve conduction positively correlated with cytokine-induced increases in frataxin expression, providing a link between increases in frataxin and neurophysiological function. Abrogation of disease-related pathology was also evident, with reductions in inflammation/gliosis and increased neural stem cell numbers in areas of tissue injury. INTERPRETATION: These experiments show that cytokines already clinically used in other conditions offer the prospect of a novel, rapidly translatable, disease-modifying, and neuroprotective treatment for Friedreich's ataxia. Ann Neurol 2017;81:212-226.

Type I interferon causes thrombotic microangiopathy by a dose-dependent toxic effect on the microvasculature.

Many drugs have been reported to cause thrombotic microangiopathy (TMA), yet evidence supporting a direct association is often weak. In particular, TMA has been reported in association with recombinant type I interferon (IFN) therapies, with recent concern regarding the use of IFN in multiple sclerosis patients. However, a causal association has yet to be demonstrated. Here, we adopt a combined clinical and experimental approach to provide evidence of such an association between type I IFN and TMA. We show that the clinical phenotype of cases referred to a national center is uniformly consistent with a direct dose-dependent drug-induced TMA. We then show that dose-dependent microvascular disease is seen in a transgenic mouse model of IFN toxicity. This includes specific microvascular pathological changes seen in patient biopsies and is dependent on transcriptional activation of the IFN response through the type I interferon α/ß receptor (IFNAR). Together our clinical and experimental findings provide evidence of a causal link between type I IFN and TMA. As such, recombinant type I IFN therapies should be stopped at the earliest stage in patients who develop this complication, with implications for risk mitigation.

Aberrant cerebellar Purkinje cell function repaired in vivo by fusion with infiltrating bone marrow-derived cells.

Bone marrow-derived cells are known to infiltrate the adult brain and fuse with cerebellar Purkinje cells. Histological observations that such heterotypic cell fusion events are substantially more frequent following cerebellar injury suggest they could have a role in the protection of mature brain neurons. To date, the possibility that cell fusion can preserve or restore the structure and function of adult brain neurons has not been directly addressed; indeed, though frequently suggested, the possibility of benefit has always been rather speculative. Here we report, for the first time, that fusion of a bone marrow-derived cell with a neuron in vivo, in the mature brain, results in the formation of a spontaneously firing neuron. Notably, we also provide evidence supporting the concept that heterotypic cell fusion acts as a biological mechanism to repair pathological changes in Purkinje cell structure and electrophysiology. We induced chronic central nervous system inflammation in chimeric mice expressing bone marrow cells tagged with enhanced green fluorescent protein. Subsequent in-depth histological analysis revealed significant Purkinje cell injury. In addition, there was an increased incidence of cell fusion between bone marrow-derived cells and Purkinje cells, revealed as enhanced green fluorescent protein-expressing binucleate heterokaryons. These fused cells resembled healthy Purkinje cells in their morphology, soma size, ability to synthesize the neurotransmitter gamma-aminobutyric acid, and synaptic innervation from neighbouring cells. Extracellular recording of spontaneous firing ex vivo revealed a shift in the predominant mode of firing of non-fused Purkinje cells in the context of cerebellar inflammation. By contrast, the firing patterns of fused Purkinje cells were the same as in healthy control cerebellum, indicating that fusion of bone marrow-derived cells with Purkinje cells mitigated the effects of cell injury on electrical activity. Together, our histological and electrophysiological results provide novel fundamental insights into physiological processes by which nerve cells are protected in adult life.

Reduced neuroprotective potential of the mesenchymal stromal cell secretome with ex vivo expansion, age and progressive multiple sclerosis.

BACKGROUND: Clinical trials using ex vivo expansion of autologous mesenchymal stromal cells (MSCs) are in progress for several neurological diseases including multiple sclerosis (MS). Given that environment alters MSC function, we examined whether in vitro expansion, increasing donor age and progressive MS affect the neuroprotective properties of the MSC secretome. METHODS: Comparative analyses of neuronal survival in the presence of MSC-conditioned medium (MSCcm) isolated from control subjects (C-MSCcm) and those with MS (MS-MSCcm) were performed following (1) trophic factor withdrawal and (2) nitric oxide-induced neurotoxicity. RESULTS: Reduced neuronal survival following trophic factor withdrawal was seen in association with increasing expansion of MSCs in vitro and MSC donor age. Controlling for these factors, there was an independent, negative effect of progressive MS. In nitric oxide neurotoxicity, MSCcm-mediated neuroprotection was reduced when C-MSCcm was isolated from higher-passage MSCs and was negatively associated with increasing MSC passage number and donor age. Furthermore, the neuroprotective effect of MSCcm was lost when MSCs were isolated from patients with MS. DISCUSSION: Our findings have significant implications for MSC-based therapy in neurodegenerative conditions, particularly for autologous MSC therapy in MS. Impaired neuroprotection mediated by the MSC secretome in progressive MS may reflect reduced reparative potential of autologous MSC-based therapy in MS and it is likely that the causes must be addressed before the full potential of MSC-based therapy is realized. Additionally, we anticipate that understanding the mechanisms responsible will contribute new insights into MS pathogenesis and may also be of wider relevance to other neurodegenerative conditions.

Reduced cellularity of bone marrow in multiple sclerosis with decreased MSC expansion potential and premature ageing in vitro.

BACKGROUND: Autologous bone-marrow-derived cells are currently employed in clinical studies of cell-based therapy in multiple sclerosis (MS) although the bone marrow microenvironment and marrow-derived cells isolated from patients with MS have not been extensively characterised. OBJECTIVES: To examine the bone marrow microenvironment and assess the proliferative potential of multipotent mesenchymal stromal cells (MSCs) in progressive MS. METHODS: Comparative phenotypic analysis of bone marrow and marrow-derived MSCs isolated from patients with progressive MS and control subjects was undertaken. RESULTS: In MS marrow, there was an interstitial infiltrate of inflammatory cells with lymphoid (predominantly T-cell) nodules although total cellularity was reduced. Controlling for age, MSCs isolated from patients with MS had reduced in vitro expansion potential as determined by population doubling time, colony-forming unit assay, and expression of ß-galactosidase. MS MSCs expressed reduced levels of Stro-1 and displayed accelerated shortening of telomere terminal restriction fragments (TRF) in vitro. CONCLUSION: Our results are consistent with reduced proliferative capacity and ex vivo premature ageing of bone-marrow-derived cells, particularly MSCs, in MS. They have significant implication for MSC-based therapies for MS and suggest that accelerated cellular ageing and senescence may contribute to the pathophysiology of progressive MS.

Cell-based therapeutic strategies for multiple sclerosis.

The availability of multiple disease-modifying medications with regulatory approval to treat multiple sclerosis illustrates the substantial progress made in therapy of the disease. However, all are only partially effective in preventing inflammatory tissue damage in the central nervous system and none directly promotes repair. Cell-based therapies, including immunoablation followed by autologous haematopoietic stem cell transplantation, mesenchymal and related stem cell transplantation, pharmacologic manipulation of endogenous stem cells to enhance their reparative capabilities, and transplantation of oligodendrocyte progenitor cells, have generated substantial interest as novel therapeutic strategies for immune modulation, neuroprotection, or repair of the damaged central nervous system in multiple sclerosis. Each approach has potential advantages but also safety concerns and unresolved questions. Moreover, clinical trials of cell-based therapies present several unique methodological and ethical issues. We summarize here the status of cell-based therapies to treat multiple sclerosis and make consensus recommendations for future research and clinical trials.

Advising patients seeking stem cell interventions for multiple sclerosis.

Given the intuitive potential of stem cell therapy and limitations of current treatment options for progressive multiple sclerosis (MS), it is not surprising that patients consider undertaking significant clinical and financial risks to access stem cell transplantation. However, while increasing evidence supports autologous haematopoietic stem cell transplantation (AHSCT) in aggressive relapsing-remitting MS, interventions employing haematopoietic or other stem cells should otherwise be considered experimental and recommended only in the context of a properly regulated clinical study. Understandably, most neurologists are unfamiliar with AHSCT procedures and the specific requirements for quality assurance and safety standards, as well as post-procedure precautions and follow-up. Consequently they may feel ill-equipped to advise patients. Here, we highlight important points for discussion in consultations with patients considering stem cell 'tourism' for MS.

Mesenchymal Stem Cell-Derived Factors Restore Function to Human Frataxin-Deficient Cells.

Friedreich's ataxia is an inherited neurological disorder characterised by mitochondrial dysfunction and increased susceptibility to oxidative stress. At present, no therapy has been shown to reduce disease progression. Strategies being trialled to treat Friedreich's ataxia include drugs that improve mitochondrial function and reduce oxidative injury. In addition, stem cells have been investigated as a potential therapeutic approach. We have used siRNA-induced knockdown of frataxin in SH-SY5Y cells as an in vitro cellular model for Friedreich's ataxia. Knockdown of frataxin protein expression to levels detected in patients with the disorder was achieved, leading to decreased cellular viability, increased susceptibility to hydrogen peroxide-induced oxidative stress, dysregulation of key anti-oxidant molecules and deficiencies in both cell proliferation and differentiation. Bone marrow stem cells are being investigated extensively as potential treatments for a wide range of neurological disorders, including Friedreich's ataxia. The potential neuroprotective effects of bone marrow-derived mesenchymal stem cells were therefore studied using our frataxin-deficient cell model. Soluble factors secreted by mesenchymal stem cells protected against cellular changes induced by frataxin deficiency, leading to restoration in frataxin levels and anti-oxidant defences, improved survival against oxidative stress and stimulated both cell proliferation and differentiation down the Schwann cell lineage. The demonstration that mesenchymal stem cell-derived factors can restore cellular homeostasis and function to frataxin-deficient cells further suggests that they may have potential therapeutic benefits for patients with Friedreich's ataxia.

Reductions in kinesin expression are associated with nitric oxide-induced axonal damage.

Axonal injury is often characterized by axonal transport defects and abnormal accumulation of intra-axonal components. Nitric oxide (NO) has a key role in mediating inflammatory axonopathy in many neurodegenerative diseases, but little is known about how nitrosative/oxidative stress affects axonal transport or whether reductions in kinesin superfamily protein (KIF) expression correlate with axon pathology. KIFs are molecular motors that have a key role in axonal and dendritic transport, and impairment of these mechanisms has been associated with a number of neurological disorders. This study shows that rat cortical neurons exposed to NO display both a time-dependent decrease in KIF gene/protein expression and neurofilament phosphorylation in addition to a reduction in axonal length and neuronal survival. Because mesenchymal stem cells (MSCs) represent a promising therapeutic candidate for neuronal/axonal repair, this study analyzes the capacity of MSCs to protect neurons and axonal transport mechanisms from NO damage. Results show that coculture of MSCs with NO-exposed neurons results in the preservation of KIF expression, axonal length, and neuronal survival. Altogether, these results suggest a potential mechanism involved in the disruption of axonal transport and abnormal accumulation of proteins in axons during nitrosative insult. We hypothesize that impaired axonal transport contributes, per se, to progression of injury and provide further evidence of the therapeutic potential of MSCs for neurodegenerative disorders.

Cell therapy for multiple sclerosis: an evolving concept with implications for other neurodegenerative diseases.

Multiple sclerosis is a major cause of neurological disability, and particularly occurs in young adults. It is characterised by conspicuous patches of damage throughout the brain and spinal cord, with loss of myelin and myelinating cells (oligodendrocytes), and damage to neurons and axons. Multiple sclerosis is incurable, but stem-cell therapy might offer valuable therapeutic potential. Efforts to develop stem-cell therapies for multiple sclerosis have been conventionally built on the principle of direct implantation of cells to replace oligodendrocytes, and therefore to regenerate myelin. Recent progress in understanding of disease processes in multiple sclerosis include observations that spontaneous myelin repair is far more widespread and successful than was previously believed, that loss of axons and neurons is more closely associated with progressive disability than is myelin loss, and that damage occurs diffusely throughout the CNS in grey and white matter, not just in discrete, isolated patches or lesions. These findings have introduced new and serious challenges that stem-cell therapy needs to overcome; the practical challenges to achieve cell replacement alone are difficult enough, but, to be useful, cell therapy for multiple sclerosis must achieve substantially more than the replacement of lost oligodendrocytes. However, parallel advances in understanding of the reparative properties of stem cells--including their distinct immunomodulatory and neuroprotective properties, interactions with resident or tissue-based stem cells, cell fusion, and neurotrophin elaboration--offer renewed hope for development of cell-based therapies. Additionally, these advances suggest avenues for translation of this approach not only for multiple sclerosis, but also for other common neurological and neurodegenerative diseases.

Cell fusion in the brain: two cells forward, one cell back.

Adult stem cell populations, notably those which reside in the bone marrow, have been shown to contribute to several neuronal cell types in the rodent and human brain. The observation that circulating bone marrow cells can migrate into the central nervous system and fuse with, in particular, cerebellar Purkinje cells has suggested, at least in part, a potential mechanism behind this process. Experimentally, the incidence of cell fusion in the brain is enhanced with age, radiation exposure, inflammation, chemotherapeutic drugs and even selective damage to the neurons themselves. The presence of cell fusion, shown by detection of increased bi-nucleated neurons, has also been described in a variety of human central nervous system diseases, including both multiple sclerosis and Alzheimer's disease. Accumulating evidence is therefore raising new questions into the biological significance of cell fusion, with the possibility that it represents an important means of cell-mediated neuroprotection or rescue of highly complex neurons that cannot be replaced in adult life. Here, we discuss the evidence behind this phenomenon in the rodent and human brain, with a focus on the subsequent research investigating the physiological mechanisms of cell fusion underlying this process. We also highlight how these studies offer new insights into endogenous neuronal repair, opening new exciting avenues for potential therapeutic interventions against neurodegeneration and brain injury.

Reductions in neuronal peroxisomes in multiple sclerosis grey matter.

BACKGROUND: Peroxisomes are organelles in eukaryotic cells with multiple functions including the detoxification of reactive oxygen species, plasmalogen synthesis and ß-oxidation of fatty acids. Recent evidence has implicated peroxisomal dysfunction in models of multiple sclerosis (MS) disease progression. OBJECTIVES: Our aims were to determine whether there are changes in peroxisomes in MS grey matter (GM) compared to control GM. METHODS: We analysed cases of MS and control GM immunocytochemically to assess peroxisomal membrane protein (PMP70) and neuronal proteins. We examined the expression of ABCD3 (the gene that encodes PMP70) in MS and control GM. Analyses of very long chain fatty acid (VLCFA) levels in GM were performed. RESULTS: PMP70 immunolabelling of neuronal somata was significantly lower in MS GM compared to control. Calibration of ABCD3 gene expression with reference to glyceraldehyde 3-phsophate dehydrogenase (GAPDH) revealed overall decreases in expression in MS compared to controls. Mean PMP70 counts in involved MS GM negatively correlated to disease duration. Elevations in C26:0 (hexacosanoic acid) were found in MS GM. CONCLUSIONS: Collectively, these observations provide evidence that there is an overall reduction in peroxisomal gene expression and peroxisomal proteins in GM neurons in MS. Changes in peroxisomal function may contribute to neuronal dysfunction and degeneration in MS.

Can reproductive health services be used to screen for sexual and gender-based violence in post-conflict Northern Uganda? - a pilot study.

Background: Sexual and gender-based violence (SGBV), including rape and child sexual abuse, remains a significant challenge in post-conflict northern Uganda. Many victims have never sought help. Consequently, the scale of the problem is not known, and SGBV victims' injuries, both psychological and physical, remain hidden and unresolved. Objectives: We aimed to explore whether health workers in rural Reproductive Health Services (RHS), following specific training, could provide a valuable resource for SGBV screening and subsequent referral to targeted services. Methods: Our project had three elements. First, RHS workers were trained to use a questionnaire to screen subjects for past SGBV Second, the screening questionnaire was used by RHS workers over a 3-month period, and the data collected were analysed to explore whether the screening approach was an effective one in this setting, and to record the scale and nature of the problem. Third, victims detected were offered referral as appropriate to hospital services or to a dedicated SGBV ActionAid shelter. Results: Of 1656 women screened, 778 (47%) had suffered SGBV: 123 rape, and 505 non-sexual violence. 1,254 (76%) had been directly or indirectly affected by conflict experiences; 1066 had lived in internally displaced persons camps. 145 (9%) requested referral to Gulu SGBV Shelter; 25 attended the shelter and received assistance, and 20 others received telephone counselling. Conclusion: Undetected SGBV remains a significant problem in post-conflict northern Uganda. RHS workers, following specific training, can effectively screen for and identify otherwise unrecognised survivors of SGBV. This matters because without ongoing detection, survivors have no opportunity for resolution, healing or help.