Congenital adrenal hyperplasia complicated by gonadotropin-dependent precocious puberty.

Precocious puberty, characterised by the early appearance of secondary sexual characteristics, poses challenges in diagnosis and management. Here, we describe a case of precocious puberty diagnosed in a boy in middle childhood, who presented with progressive phallus enlargement, pubic hair development and increased aggressive behaviour. Hormonal evaluation confirmed the diagnosis of congenital adrenal hyperplasia (CAH), complicated by gonadotropin-dependent precocious puberty. The case highlights the importance of assessment of testicular volume in a patient presenting with precocious puberty. Symmetrical testicular enlargement in a patient with CAH suggests premature activation of the hypothalamic-pituitary-gonadal axis. The patient received glucocorticoid therapy to suppress androgen production related to CAH and gonadotropin-releasing hormone analogue therapy to control premature activation of the hypothalamic-pituitary-gonadal axis. Follow-up visits showed regression of secondary sexual characteristics and improved growth velocity.

Patterns and outcomes of late onset thyroid disturbances after COVID-19 vaccination: A report of 75 cases.

Isolated cases of subacute thyroiditis exist in the early period of COVID-19 vaccination, largely after mRNA vaccines. Here we report late onset thyroid disturbances and persistent health issues in patients of thyroid disorders after COVID-19 vaccination. Seventy-five patients with post COVID-19 vaccination thyroid disturbances were identified. Among these, 41 had flare of underlying thyroid illness, majority occurring at a median time lag of 28.4 weeks since 2nd dose. Thirty-one cases of new onset hypothyroidism and three of new onset hyperthyroidism were reported, with a median time lag respectively of 17.2 and 22.6 weeks since 2nd dose. Most cases occurred after ChAdOx1-nCoV-19, which was the commonest vaccine employed in mass roll out in India. Significant improvement was observed in majority, after a median follow up of 22-26 weeks. New onset health issues persisting for ≥4 weeks were reported in 37.3% and were common in individuals with history of COVID-19 before vaccine. New onset metabolic, musculoskeletal, and reproductive disorders were the common health complaints. Active monitoring is warranted for late onset adverse events after COVID-19 vaccines of all types. Larger studies with involvement of unvaccinated individuals are required to understand the incidence and causality of late onset thyroid disturbances after COVID-19 vaccines.

Heterogeneity in Disulfide Bond Reduction in IgG1 Antibodies Is Governed by Solvent Accessibility of the Cysteines.

We studied unpaired cysteine levels and disulfide bond susceptibility in four different γ-immunoglobulin antibodies using liquid chromatography-mass spectrometry. Our choice of differential alkylating agents ensures that the differential peaks are non-overlapping, thus allowing us to accurately quantify free cysteine levels. For each cysteine residue, we observed no more than 5% to be unpaired, and the free cysteine levels across antibodies were slightly higher in those containing lambda light chains. Interchain and hinge residues were highly susceptible to reducing stresses and showed a 100-1000-fold higher rate of reduction compared to intrachain cysteines. Estimations of the solvent-accessible surface for individual cysteines in IgG1, using an implicit all-atom molecular dynamics simulation, show that interchain and hinge cysteines have >1000-fold higher solvent accessibility compared to intrachain cysteines. Further analyses show that solvent accessibility and the rate of reduction are linearly correlated. Our work clearly establishes the fact that a cysteine's accessibility to the surrounding solvent is one of the primary determinants of its disulfide bond stability.

The Prevalence of Anemia in Hospitalized Patients With Diabetic Foot Ulcer (DFU) and the Relationship Between the Severity of Anemia and the Severity of DFU.

BACKGROUND AND AIMS: We aim to determine the prevalence of anemia in hospitalized patients with diabetic foot ulcers (DFUs) and estimate the relationship between the severity of anemia and diabetic foot ulcer. MATERIALS AND METHODS: We retrospectively collected and evaluated the data of 323 patients hospitalized with diabetic foot ulcer (DFU). We included 299 type 2 diabetic patients with foot ulcers of neuropathic or neuroischemic nature with infection. Anemia was defined based on World Health Organization (WHO) criteria, and the severity of DFU was classified in University of Texas (UT) grades. RESULTS: Anemia was detected in 94.3% of DFU, and the prevalence of mild, moderate, and severe anemia was 16.7%, 55.7%, and 27.6%, respectively. There was a significant difference in the mean hemoglobin (Hb) levels among the patients with varying grades of severity of DFU (1B: Hb=10.17±2.08 gm/dL, 2B: Hb=9.27±2.04 gm/dL, 3B: Hb=8.03±1.829 gm/dL; p value=<0.0001). The iron study was available in 141 (47.15%) patients and was suggestive of anemia of chronic disorder (mean serum iron=40.22±23.81 mcg/dL, mean total iron-binding capacity (TIBC)=239.34±67.24 mcg/dL, mean ferritin=378.05±141.337 ng/mL). TIBC significantly decreased (1B=262.13±61.05, 2B=233.65±71.26, 3B=222.43±74.18; p=0.04), and ferritin significantly increased (1B=309.9±70.76, 2B=351.73±94.22, 3B=488.58±170.4; p<0.0001) with increasing DFU severity. Hemoglobin was significantly decreased at the time of discharge in comparison to that at admission (9.3±2.1 gm/dL versus 8.8±1.5 gm/dL; p value=0.01). Red blood cell (RBC) counts, mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), lymphocyte counts, albumin, calcium, and high-density lipoprotein (HDL) significantly decreased with the increase of DFU severity. The duration of hospitalization, total leucocyte counts, neutrophil counts, and neutrophil-to-lymphocyte ratio (NLR) increased with the severity of DFU. CONCLUSIONS: The prevalence of anemia was very high in DFU and more than three-fourths of the patients had moderate to severe anemia. The severity of anemia was associated with the severity of DFU. The most common cause of anemia was anemia of chronic disorder secondary to diabetic foot infection. During the period of hospitalization, hemoglobin decreased despite improvement in DFU infection.

IgG1 and IgG4 antibodies sample initial structure dependent local conformational states and exhibit non-identical Fab dynamics.

We have investigated the dynamics of two [Formula: see text]-immunoglobulin molecules, IgG1 and IgG4, using long all atom molecular dynamics simulations. We first show that the de novo structures of IgG1 and IgG4 predicted using AlphaFold, with no interactions between the fragment crystallizable (Fc) domain and the antigen fragment binding domain (Fab), eventually relaxes to a state with persistent Fc-Fab interactions that mirrors experimentally resolved structures. We quantified the conformational space sampled by antibody trajectories spawned from six different initial structures and show that the individual trajectories only sample states bound by a local minimum and display very little mixing in their conformational states. Furthermore, the dynamics of the individual Fab domains are strongly dependent on the initial crystal structure and isotype. In all conditions, we observe non-identical dynamics between the Fab arms in an antibody. For a six-bead coarse grained model, we show that non-covalent Fc-Fab interactions can modulate the stiffnesses associated with Fc-Fab distances, angles, and dihedral angles by up to three orders of magnitude. Our results clearly illustrate the inherent complexities in studying antibody dynamics and highlight the need to include non-identical Fab dynamics as an inherent feature in computational models of therapeutic antibodies.

Histological Spectrum of Clinical Kidney Disease in Type 2 Diabetes Mellitus Patients with special Reference to nonalbuminuric Diabetic Nephropathy: A Kidney Biopsy-based Study.

BACKGROUND: Diabetic nephropathy (DN) is an important and catastrophic complication of diabetes mellitus (DM). Kidney disease has heterogeneity in histology in diabetes patients and includes both diabetic kidney disease (DKD) (albuminuric or nonalbuminuric) and nondiabetic kidney disease (NDKD) either in isolation or in coexistence with DN. Diabetic nephropathy is hard to overturn. While NDKD is treatable and reversible. MATERIALS AND METHODS: We enrolled a total of 50 type 2 diabetes mellitus (T2DM) patients with clinical kidney disease, of both genders and age >18 years, who underwent kidney biopsy from October 2016 to October 2018. Patients with proteinuria <30 mg per day were excluded from the study. The indications of the renal biopsy were nephrotic syndrome (NS), active urinary sediment, rapid decline in renal function, asymptomatic proteinuria, and hematuria. RESULT: A total of 50 (males: 42 and females: eight) patients with T2DM who underwent kidney biopsy were enrolled. The clinical presentation was: NS 26 (52%), chronic kidney disease (CKD) 11 (22%), asymptomatic proteinuria and hematuria six (12%), acute kidney injury (AKI) four (8%), and acute nephritic syndrome (ANS) three (6%). Diabetic retinopathy (DR) was noted in 19 (38%) cases. Kidney biopsy revealed isolated DN, isolated NDKD, and NDKD superimposed on DN in 26 (52%), 14 (28%), and 10 (20%) cases, respectively. Idiopathic membranous nephropathy (MN) (4) and amyloidosis (2) were the most common forms of NDKD, whereas diffuse proliferative glomerulonephritis (DPGN) was the main form of NDKD superimposed on DN. Diabetic nephropathy was observed in 15 (79%) cases in presence of DR and also in 11 (35.5%) cases even in absence of DR. Of eight patients with microalbuminuria four (50%) cases have biopsy-proven DN. CONCLUSION: About 48% of patients had NDKD either in isolation or in coexistence with DN. Diabetic nephropathy was found in absence of DR and in patients with a low level of proteinuria. The level of proteinuria and presence of DR does not help to distinguish DN vs NDKD. Hence, renal biopsy may be useful in selected T2DM patients with clinical kidney disease to diagnose NDKD.

COVID-19-associated Mucormycosis: A clinico-epidemiological study.

BACKGROUND: There was an unprecedented increase in COVID-19-associated-Mucormycosis (CAM) cases during the second pandemic wave in India. METHODS: This observational study was done to know the epidemiological profile of CAM cases andincluded all patients admitted with mucormycosis between May 2021 and July 2021. RESULTS: Out of the enrolled 208 CAM cases (either SARS-CoV-2 RT-PCR or serology positive), 204, three and one had rhino-orbital-cerebral, pulmonary and gastrointestinal mucormycosis, respectively. 95.7 % of the patients had diabetes, out of which 42.3 % were recently diagnosed. Mean HbA1c was 10.16 ± 2.56 %. 82.5 % of the patients were unvaccinated. During their COVID-19 illness, 86.5 % were prescribed antibiotics, 84.6 % zinc preparations, 76.4 % ivermectin, and 64.9 % steroids, while only 39.5 % required oxygen therapy. The frequency of blood groups A, B, O and AB in our CAM patients was 29.5 %, 18.9 %, 38.9 % &12.6 %, respectively. At three months follow up, 60 (28.8 %) patients died, four (1.9 %) stopped antifungal treatment, and 144(69.23 %) were on antifungal treatment. 55 % (n = 33) of deaths occurred within 15 days of admission. Mortality was significantly associated with higher age, RT-PCR positive for SARS-CoV-2, raised serum creatinine and alkaline phosphatase during treatment. At 6 months follow-up, eight more patients died, three due to chronic kidney disease, four patients who had stopped treatment and one patient who was on a ventilator due to COVID-19 associated pneumonia and the rest 140(67.3 %) survived. CONCLUSION: Uncontrolled hyperglycemia, SARS-CoV-2 infection, rampant use of antibiotics, zinc supplementation and steroids were some of the risk factors for mucormycosis. Despite the overwhelming number of patients with an uncommon disease like mucormycosis, the six months mortality was much lower than expected.

Association of Variant rs7903146(c/t) Single Nucleotide Polymorphism of Transcription Factor 7-like 2 Gene with Newly Detected Hyperglycemia in Pregnancy.

Background: Transcription factor 7-like 2 (TCF7L2) gene has a significant role in hyperglycemia in pregnancy (HIP) risk. The current study was planned with the aim to evaluate the association of single nucleotide polymorphism (SNP) rs7903146 in patients of newly detected HIP among Indian population of northern region. Methods: This study was an observational case control study done among newly detected HIP (The World Health Organization (WHO) criteria, 2013) and healthy pregnant females without diabetes. Participants from both the group were genotyped for rs7903146 (C/T) variant of TCF7L2 gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Results: A total of 71 cases of newly detected HIP were included in the study, out of which 25 (35.2%) of them were of first-time detected diabetes mellitus in pregnancy (DIP) and 46 (64.7%) were of gestational diabetes (GDM) and 100 were pregnant females without diabetes in third trimester were enrolled as controls. Average age of participants in the case group was 28.7 ± 4.0 years and the control group were 26.5 ± 3.6 years (P value 0.09). The wild homozygous CC genotype, heterozygous CT genotype and homozygous TT genotype were present in 39.4%, 53.5%, 7.1% of case group vs 53%, 43% and 4% of control group, respectively. No significant association of rs7903146(C/T) SNP of TCF7L2 gene in HIP (CC/CT, CC/TT P value 0.15, 0.38, respectively) in our population was found. There was no significant difference in the distribution of genotypes between DIP and GDM. Conclusion: This study shows no evidence of association of rs7903146(C/T) SNP of TCF7L2 gene with newly detected HIP in our population.

Non-covalent Fc-Fab interactions significantly alter internal dynamics of an IgG1 antibody.

The fragment-antigen-binding arms (Fab1 and Fab2) in a canonical immunoglobulin G (IgG) molecule have identical sequences and hence are always expected to exhibit symmetric conformations and dynamics. Using long all atom molecular simulations of a human IgG1 crystal structure 1HZH, we demonstrate that the translational and rotational dynamics of Fab1 and Fab2 also strongly depend on their interactions with each other and with the fragment-crystallizable (Fc) region. We show that the Fab2 arm in the 1HZH structure is non-covalently bound to the Fc region via long-lived hydrogen bonds, involving its light chain and both heavy chains of the Fc region. These highly stable interactions stabilize non-trivial conformer states with constrained fluctuations. We observe subtle modifications in Fab1 dynamics in response to Fab2-Fc interactions that points to novel allosteric interactions between the Fab arms. These results yield novel insights into the inter- and intra-fragment motions of immunoglobulins which could help us better understand the relation between their structure and function.

Clinical Prediction of Type 2 Diabetes Mellitus (T2DM) via Anthropometric and Biochemical Variations in Prakriti.

Type 2 Diabetes Mellitus (T2DM) is a complicated multifactorial illness involving hereditary and external environmental variables. The symptoms typically appear gradually over a number of years without realizing it. This viewpoint is further supported by the Ayurvedic constitution concept (Prakriti). Prakriti explains the biological variability that is observed in different individuals. This study was conducted a retrospective investigation to examine if there was a link between type 2 diabetes and an individual's constitution based on anthropometric and biochemical characteristics. Physical and mental characteristics and anthropometric and biochemical markers were used to determine reported cases' prevailing Dosha Prakriti (constitution). Based on biochemical and anthropometric data, significant differences in Prakriti were found between the case (T2DM patients) and control (person without diabetes) groups. The incidence of numerous secondary problems linked with T2DM patients was also evaluated according to their Prakriti types, which revealed a positive relationship. The three primary contributing parameters, such as waist-hip ratio, postprandial blood sugar, and serum creatinine, were correctly classified all person with or without diabetes subjects to 90.6% of the time, whereas the constitution-wise study classified person with diabetes and without diabetes individuals of Pitta and Kapha Prakriti to 94.3% and 90%, respectively. A discriminant function was created to predict a person with diabetes and without diabetes based on these three contributing factors. The primary contributing biochemical parameters discovered by Prakriti in the current study could be used as a biochemical disease diagnostic for predicting type 2 diabetes susceptibility.

Revisiting Rhombic Flaps for Aesthetic Facial Resurfacing: Addressing a Surgical Conundrum.

BACKGROUND: Facial resurfacing is a surgical and aesthetic challenge, as it requires soft, pliable, and stretchable donor skin with a good match of color, texture, and thickness and minimum donor scarring. Rhombic flap is a highly versatile flap that has the aforementioned properties. Careful flap planning and execution is vital for successful outcomes. AIMS: The aim of this work was to study the geometry of the classic rhombic flaps; to evaluate their versatility and technical finesse as well as their application to resurface defects over various regions of the face. MATERIALS AND METHODS: Overall, 42 patients with facial scars due to trauma, malignancies, small nevi, and mature scars of the face were studied and operated on. Of the four rhombic flaps that were possible, choosing the best option required careful attention to camouflage of scar and proximity to vital deformable anatomical structures. Geometrical planning of the flap was the essence of the entire surgical exercise. RESULTS: Outcome of rhombic flaps were meticulously and critically analyzed. The study consisted of 36 females (85.71%) and only 6 males with a mean age of 23 years. Defects were created on 27 mature scars (64.29%) and 11 burn scars (26.19%). Defects over the cheek and lateral canthus of the eye were especially problematic. It is a safe flap with excellent results as far as visible scarring and traction deformities are concerned. Scars elsewhere on the face fared better than on the bony prominence. CONCLUSION AND SIGNIFICANCE: Rhombic flaps offer an excellent alternative to resurface facial scars and defects in a selected subset of patients.

Radiological Evaluation of Postsurgical Course of Perforators in Retrograde Posterior Tibial Fasciocutaneous Flaps for Distal Leg and Foot Defects.

Background Mapping of vascular perforators by various methodologies have been described for planning of a variety of flaps in the lower limbs. We attempted to assess the changes in posterior tibial perforators after transfer of fasciocutaneous flaps for leg defects. Methods 20 patients with distal leg and foot defects were studied by computed tomography angiography (CTA) and preoperative audio Doppler to ascertain perforators of posterior tibial artery. Fasciocutaneous flaps were raised, based on these perforators, depending on the site and size of soft-tissue defects. The number of perforators and their distance from the medial malleolus were also studied. Postoperative CTA was performed on the 7th to 10th day, with emphasis on postoperative changes of the perforators on which the flaps were based. Results One to four posterior tibial perforators were found between 5 cm and 8 cm proximal to the medial malleolus. After flap transfer, the perforators could be traced to variable distance through the total length of the flap. The perforators formed small vascular loop in 12 patients, following retrograde posterior tibial flap transfer. The height of the loop, the number of such loops, the dilatation and tortuosity of the perforators, and their longitudinal orientation were studied in detail. Most of the findings can be explained by mechanical realignment of perforators as well as by the delay phenomenon associated with retrograde fasciocutaneous flaps. Conclusion It was concluded that the morphological changes associated with the perforators explained the vascular rationality and success of these flaps.

Diabetes and rhino-orbito-cerebral mucormycosis - A deadly duo.

BACKGROUND: Rhino-orbito-cerebral mucormycosis(ROCM) is an uncommon yet potentially fatal fungal infection predominantly seen in immunocompromised individuals. However, there is very limited data available from India regarding outcome of patients with ROCM and diabetes mellitus. OBJECTIVE: To ascertain clinical parameters and factors in the final outcome of patients with diabetes mellitus and ROCM. MATERIALS AND METHODS: This series included retrospective analysis of medical records of 91 patients with diabetes mellitus who were diagnosed with ROCM from january 2007 to june 2019 at a tertiary care hospital in Punjab. RESULTS: The mean age of patients was 52.6 years (range 18-82 years), with men constituting the majority (71.4 %). Ophthalmoplegia was the most frequent presenting feature seen in 77 % of patients followed by proptosis (71 %). Intracranial involvement was seen in 20 % of the patients and cavernous sinus thrombosis was diagnosed in 9(10 %) patients. Out of 91 patients, 81 patients were subjected to appropriate surgical procedure depending upon site and extent of involvement by mucorales. A total of 53 (58.2 %) patients survived while 38(41.8 %) patients succumbed. Delay in presentation to hospital, intracranial extension and loss of vision at presentation adversely affected the outcome (p < 0.05). Aggressive surgical management in the form of multiple debridements was superior to single debridement (p < 0.05). Diabetic ketoacidosis did not significantly affect the outcome (p = 0.359). CONCLUSIONS: ROCM in patients with diabetes mellitus, is a rapidly progressive disease with a high fatality rate and grave outcome unless diagnosed early and managed aggressively.

Early detection of cutaneous complications of insulin therapy in type 1 and type 2 diabetes mellitus.

BACKGROUND: Subcutaneous insulin therapy is associated with important injection site complications, which can influence insulin pharmacokinetics resulting in glycemic fluctuations above and below target levels for blood glucose. OBJECTIVE: Our objective was to assess the prevalence and risk factors of cutaneous complications including insulin derived amyloidosis in insulin-injecting diabetes patients and to study the role of ultrasonography (in comparison to gel-assisted palpation) in early diagnosis of lipohypertrophy (LH). METHODS: This was a cross-sectional study conducted at a tertiary care center in India, wherein 500 patients injecting insulin for ≥2 years were randomly enrolled and evaluated for the presence of cutaneous complications of insulin therapy through clinical examination, ultrasonography and punch biopsy of skin. RESULTS: Clinical examination detected LH in 44.6% of patients. Ultrasonography diagnosed additional 13.4% of patients with LH which were missed on clinical examination. Incorrect rotation of sites (P < 0.001) and insulin syringe reusage for more than five times (P < 0.001) significantly increased the risk of LH. Skin biopsy was performed in 100 cases, out of which two patients showed apple green birefringence and its association with insulin was confirmed by positive staining with anti insulin antibody in these two patients. CONCLUSION: Improper rotation of sites and reuse of needles were the leading causes of LH in Indian diabetic patients. Ultrasonography is more objective and reliable method of detecting LH. Insulin-derived amyloidosis may be a more common complication of insulin therapy than previously thought.

Predictors of the diets consumed by adolescent girls, pregnant women and mothers with children under age two years in rural eastern India.

Adolescents, pregnant women and mothers of children under 2 years of age are in stages of life characterized by higher nutritional demands. The study measured the dietary diversity of 17,680 adolescent girls, pregnant women and mothers of children under age 2 years in the eastern Indian states of Bihar, Chhattisgarh and Odisha using data from the Swabhimaan baseline survey conducted in 2016. The association of women's mean Dietary Diversity Scores with socioeconomic, health and nutrition service indicators was assessed. The sampled population was socioeconomically more vulnerable than the average Indian population. There was not much variation in the types of foods consumed daily across target groups, with diet being predominantly cereal (98%) and vegetable (83%) based. Nearly 30% of the mothers had low Dietary Diversity Scores, compared with 25% of pregnant women and 24% of adolescent girls. In each target group, more than half of the respondents were unable to meet the Minimum Dietary Diversity score of at least five of ten food groups consumed daily. Irrespective of their background characteristics, mean Dietary Diversity Scores were significantly lower in Bihar than in Chhattisgarh and Odisha for all target groups. Having at least 6 years of education, belonging to a relatively rich household and possessing a ration card predicted mean dietary diversity. Project interventions of participatory women's group meetings improved mean Dietary Diversity Scores for mothers and adolescent girls. Considering the association between poverty and dietary diversity, the linkage between girls and women and nutrition-focused livelihoods and supplementary nutrition programmes needs to be tested.

Evaluating the effect of strengthening nurse midwifery pre-service education in two Indian states: A single group pre - and post - intervention study.

BACKGROUND: The high focus states of India have higher maternal and neonatal mortality rates as compared to the national average. The quality of pre-service education (PSE) in nursing midwifery institutions in these states was found to be suboptimal. In 2013, Government of India implemented the PSE strengthening program across all public sector nursing midwifery institutions. The program focused on strengthening educational processes, training infrastructure, institutional management and clinical site practices by implementing a set of performance standards. OBJECTIVE: To evaluate the effect of PSE strengthening program on institutional performance and maternal neonatal health (MNH) related competences of nursing midwifery students and faculty. DESIGN: Single group pre-post intervention study. SETTINGS: 15 sampled public sector nursing midwifery institutions in the states of Madhya Pradesh and Odisha, India. PARTICIPANTS: Final-year students and faculty involved in teaching MNH subjects. METHODS: Performance of 15 sampled institutions was assessed at baseline, midline and endline using the performance standards. Additionally, competences of 232, 295 and 298 students and 64, 62 and 63 faculty members at baseline, midline and endline respectively were assessed on six MNH related practices using objective structured clinical examination (OSCE). RESULTS: None of the institutions met 70% standards during baseline. At endline, 13 of the 15 institutions met these standards. The mean OSCE scores of students and faculty at baseline was 17.1 (SD: 8.0) and 23.5 (SD: 14.3) out of 76 respectively. It significantly increased to 66.4 (SD: 8.0) and 71.1 (SD: 5.2) during the endline. The proportion of students and faculty found competent (achieved 75% in OSCE) also significantly increased from none at baseline to 91% and 98% respectively, at endline. CONCLUSION: The combination of attributes included in the PSE strengthening program may have contributed to improvements in institutional performance as well as MNH related competences of nursing midwifery students and faculty.

Tryptophan Oxidation of a Monoclonal Antibody Under Diverse Oxidative Stress Conditions: Distinct Oxidative Pathways Favor Specific Tryptophan Residues.

Tryptophan oxidation can play an important role in selecting therapeutic monoclonal antibodies for commercialization. Monoclonal antibodies that harbor particularly sensitive tryptophan residues are typically discarded in favor of oxidation resistant antibodies. The susceptibility of any individual tryptophan residue to oxidation is typically evaluated through forced degradation studies during the molecule development process. We compared the results of several common forced degradation "stress tests" for each tryptophan residue in a monoclonal antibody and found that high-stress oxidation conditions consistently provide a different ranking of oxidative sensitivity across the individual tryptophan residues compared to long-term thermal stability or low-stress conditions. We subsequently determined that this difference in ranking is largely due to an overabundance of double oxidation (i.e. detected as +32 Da) of specific tryptophan residues under high stress conditions compared to single oxidation (i.e. +16 Da). We posit that this double oxidation is in fact mechanistically distinct from the observed single oxidation and that high stress conditions favor the double oxidation mechanism (and double oxidation sensitive tryptophan residues) while single oxidation appears to be the primary mode of oxidation under H2O2 stress and long-term thermal stability and favors different tryptophan residues which are more susceptible to the single oxidation mechanism.

Engineered IL-21 Cytokine Muteins Fused to Anti-PD-1 Antibodies Can Improve CD8+ T Cell Function and Anti-tumor Immunity.

Inhibitors that block the programmed cell death-1 (PD-1) pathway can potentiate endogenous antitumor immunity and have markedly improved cancer survival rates across a broad range of indications. However, these treatments work for only a minority of patients. The efficacy of anti-PD-1 inhibitors may be extended by cytokines, however, the incorporation of cytokines into therapeutic regimens has significant challenges. In their natural form when administered as recombinant proteins, cytokine treatments are often associated with low response rates. Most cytokines have a short half-life which limits their exposure and efficacy. In addition, cytokines can activate counterregulatory pathways, in the case of immune-potentiating cytokines this can lead to immune suppression and thereby diminish their potential efficacy. Improving the drug-like properties of natural cytokines using protein engineering can yield synthetic cytokines with improved bioavailability and tissue targeting, allowing for enhanced efficacy and reduced off-target effects. Using structure guided engineering we have designed a novel class of antibody-cytokine fusion proteins consisting of a PD-1 targeting antibody fused together with an interleukin-21 (IL-21) cytokine mutein. Our bifunctional fusion proteins can block PD-1/programmed death-ligand 1 (PD-L1) interaction whilst simultaneously delivering IL-21 cytokine to PD-1 expressing T cells. Targeted delivery of IL-21 can improve T cell function in a manner that is superior to anti-PD-1 monotherapy. Fusion of engineered IL-21 variants to anti-PD1 antibodies can improve the drug-like properties of IL-21 cytokine leading to improved cytokine serum half-life allowing for less frequent dosing. In addition, we show that targeted delivery of IL-21 can minimize any potential detrimental effect on local antigen-presenting cells. A highly attenuated IL-21 mutein variant (R9E:R76A) fused to a PD-1 antibody provides protection in a humanized mouse model of cancer that is refractory to anti-PD-1 monotherapy. Collectively, our preclinical data demonstrate that this approach may improve upon and extend the utility of anti-PD-1 therapeutics currently in the clinic.

Association of Acanthosis Nigricans and Acrochordon with Insulin Resistance: A Cross-Sectional Hospital-Based Study from North India.

BACKGROUND: Insulin resistance (IR) is a pre-diabetic condition and has been reported in patients with acanthosis nigricans (AN) and acrochordon. AN and acrochordon are claimed to be cutaneous markers of IR. AIM: The purpose of this paper was to study the association of AN and acrochordon with IR. METHODS: It was a cross-sectional hospital-based study. Both groups were assessed for IR by using homeostatic model assessment of insulin resistance (HOMA-IR) formula. RESULTS: A total of 70 cases and an equal number of controls were studied. IR was observed more in cases (41.4%) compared to controls (17.1%) (P < 0.01). Mean HOMA-IR value was also significantly higher in cases (4.32 ± 4.44) compared to controls (2.27 ± 0.90) (P < 0.05). LIMITATIONS: Low number of cases and controls were taken in the study. Association with hyperlipidemia and metabolic syndrome was not elicited. CONCLUSIONS: AN and acrochordons were found to be associated with IR.

Effects of Buffer Composition on Site-Specific Glycation of Lysine Residues in Monoclonal Antibodies.

Candidate antibodies under consideration for development as pharmaceuticals must be screened for potential liabilities. Glycation of lysine side chains is one liability which can significantly alter the efficacy of a therapeutic antibody. Antibody candidates are often subjected to stress-testing after purification to assess liabilities that may arise from variability in the manufacturing process and gauge the manufacturability of the molecule. Because previous publications have shown significant site-specific effects of certain buffer components on the glycation rate of individual lysines, we sought to understand the effects of common buffering agents to find suitable buffers for glycation stress-testing (forced glycation). Therapeutic antibodies are typically only exposed to reducing sugars in cell culture media during production, so we sought to identify buffers that could be used as surrogates for media in forced glycation reactions. Our results indicate that common buffering agents can drastically alter the rate of glycation for specific lysines in an antibody. Forced glycation reactions performed in HEPES and citrate buffers both produce site-specific lysine glycation rates that correlate well with cell culture media, whereas bicarbonate buffer has a highly stimulatory effect on most lysines leading to higher total glycation levels and a poor correlation to glycation rates in media.